Determining WT-1-Specific T Cells and WT-1 Specific T Cell Receptors (TCRS)

ABSTRACT

The invention is directed to methods for determining antigen-specific T cells generally and to T cell receptors which bind an epitope of the Wilms&#39; tumor antigen-1 (WT1) protein specifically. The disclosure also provides polynucleotides encoding the TCRs, engineered cells exogenously expressing the TCRs, and methods of making and using the TCRs and/or cells expressing the TCRs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of and claims priority toU.S. patent application Ser. No. 15/827,639, filed Nov. 30, 2017, whichis in turn a continuation of U.S. patent application Ser. No.14/242,520, filed Apr. 1, 2014. This application also claims priority toU.S. Provisional Patent Application No. 62/519,088, filed Jun. 13, 2017,and U.S. Provisional Patent Application No. 62/629,496, filed Feb. 12,2018. The disclosures of each of these referenced applications arehereby incorporated by reference in their entireties.

REFERENCE TO THE ELECTRONIC TEXT FILE SUBMITTED HEREWITH

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:ADBS_079_02US_SeqList_ST25.txt; date recorded: Jun. 11, 2018; file size:55.2 kilobytes).

BACKGROUND OF THE INVENTION

Many crucial immune functions are mediated by T cell receptors (TCRs),which comprise α and β subunits that together bind to a complexconsisting of an antigenic peptide and major histocompatibility complex(MHC) molecules. It is believed that several important diseases arisefrom aberrant T cell function: For example, cancers are thought to arisefrom a failure of immune surveillance, that is, the T cell function ofdetecting and destroying clones of transformed cells before they growinto tumors; and autoimmune diseases are thought to arise from an overactive or aberrant response of T cells to self-antigens (Abbas et al,Cellular and Molecular Immunology, Fourth Edition (W. B. SaundersCompany, 2000)). Consequently, there has been interest both inidentifying and tracking antigen-specific T cells and in harnessing Tcell functions in several therapeutic approaches for the treatment bothcancer and autoimmune diseases, e.g. Molloy et al, Current Opinion inPharmacology, 5: 438-443 (2005); Morgan et al, Science, 314: 126-129(2006); Turcotte and Rosenberg, Adv. Surg., 45: 341-360 (2011). Severalchallenges are posed by these interests: Current techniques foridentifying and tracking antigen-specific T cells, especially on a largescale, are difficult and expensive, and likewise, current techniques foridentifying and isolating paired TCRα and TCRβ subunits that form afunctional receptor are difficult and expensive. In regard to detectingantigen-specific T cells, the use of direct multimer staining requireslaborious development of specific HLA-restricted reagents, and otherassays, such as ELISPOT, intracellular cytokine staining, andproliferation assays, enumerate antigen-specific T cells based ondetection of activation following stimulation of the T cells in vitrowith antigen, e.g. Gratama et al, Cytometry A, 73A: 971-974 (2008). Inregard to isolating functional pairs of TCR chains, typically a T cellof interest is identified and clonally expanded to enable isolation andanalysis of nucleic acids encoding each subunit. Even for a commondisease antigen, such as MART-1 in melanoma, the process of single cellanalysis, cloning and receptor isolation must be repeated for eachpatient.

Recently, diagnostic and prognostic applications have been proposed thatuse large-scale DNA sequencing as the per-base cost of DNA sequencinghas dropped and sequencing techniques have become more convenient, e.g.Welch et al, Hematology Am. Soc. Hematol. Educ. Program, 2011: 30-35;Cronin et al, Biomark Med., 5: 293-305 (2011); Palomaki et al, Geneticsin Medicine (online publication 2 Feb. 2012). In particular, profiles ofnucleic acids encoding immune molecules, such as T cell or B cellreceptors, or their components, contain a wealth of information on thestate of health or disease of an organism, so that diagnostic andprognostic indicators based on the use of such profiles are beingdeveloped for a wide variety of conditions, Faham and Willis, U.S.Patent Application Publication No. 2010/0151471; Freeman et al, GenomeResearch, 19: 1817-1824 (2009); Boyd et al, Sci. Transl. Med., 1(12):12ra23 (2009); He et al, Oncotarget (Mar. 8, 2011). Currentsequence-based profiles of immune repertoires consist of nucleic acidsencoding only single receptor chains; thus, potentially usefulinformation from correctly paired TCRα and TCRβ chains is not available.

Wilms' tumor oncogene protein (WT1) is a zinc finger transcriptionfactor involved in the development of the urogenital system and isexpressed in a variety of cancers. After birth, WT1 expression islimited to low expression in only a few cell types in normal tissues;however, in certain cancers, WT1 is overexpressed and associated withcancer cell survival and proliferation. Aberrant and/or overexpressionof WT1 has been observed in Wilms' tumors, leukemias, and other cancers.Therefore, WT1 is an attractive target for immunotherapy.

SUMMARY OF THE INVENTION

In view of the above, it would be highly useful for cancer, infectiousdisease, and autoimmune disease treatment if there were availableconvenient methods for determining functional, antigen-specific, immunereceptors from nucleic acids encoding subunits of the immune receptorsthat have been separately extracted and sequenced. As such, the presentdisclosure provides methods for determining functional,antigen-specific, immune receptors, in particular T cell receptors(TCRs), from nucleic acids encoding subunits of the immune receptors(e.g., TCR alpha and TCR beta chains) that have been separatelyextracted and sequenced. The present disclosure further provides TCRsspecific for the WT1 oncoprotein and nucleic acid encoding the same,cells comprising such TCRs, methods of use, and methods of identifyingand producing such TCRs. In some embodiments, the present disclosureprovides one or both of the alpha (α) chain and the beta (β) chain ofantigen-specific TCRs (e.g., WT1-specific TCR alpha and/or beta chains),and nucleic acids encoding these alpha and beta chains. In someembodiments, the present disclosure provides TCRs and T cellsexogenously expressing TCRs specific for WT1 oncoprotein that are usefulin therapeutic and/or diagnostic methods for WT1-expressing cancers.These and other aspects of the invention are exemplified in a number ofimplementations and applications described throughout the specificationand summarized below.

In some embodiments, the present disclosure provides methods fordetermining antigen-specific T cells in a tissue sample comprising thefollowing steps: (a) reacting a tissue sample comprising T cells with anantigen under activation conditions in a reaction mixture; (b) sorting Tcells from the reaction mixture into a subset of antigen-specific Tcells and/or activated T cells and a subset of non-antigen-specific Tcells and/or un-activated T cells; (c) sequencing recombined nucleicacids encoding a T-cell receptor (TCR) chain or a portion thereof fromthe antigen-specific T cells and/or activated T cells to providesequence reads from which clonotypes are determined; (d) sequencingrecombined nucleic acids encoding a TCR chain or a portion thereof fromthe non-antigen-specific T cells and/or un-activated T cells to providesequence reads from which clonotypes are determined; and (e) determiningantigen-specific T cells in the tissue sample as T cells whose clonotypefrequencies increase in the subset of sorted antigen-specific and/oractivated T cells relative to the frequencies of the same clonotypes inthe subset of sorted non-antigen-specific and/or un-activated T cells.

In some embodiments, the present disclosure provides methods ofdetermining antigen-specific T cell receptors from a sample containing Tcells comprising the following steps: (a) sequencing recombined nucleicacids encoding one or more TCR chain(s), or a portion thereof, from afirst portion of the sample to generate a first multiplicity (numbergreater than 2) of sequence reads obtained from un-stimulated T cells;(b) partitioning a second portion of the sample into a plurality (numberequal or greater than 2) of reaction mixtures and exposing each reactionmixture of the plurality of reaction mixtures to antigens; (c) for eachreaction mixture in the plurality of reaction mixtures, separating Tcells that interact with one or more antigens in the reaction mixture toobtain a subset of antigen-specific T cells, wherein each of the subsetsof antigen-specific T cells corresponds to one reaction mixture in theplurality of reaction mixtures; (d) for each of the subsets ofantigen-specific T cells separated in step (c), sequencing recombinednucleic acids encoding one or more TCR chain(s), or a portion thereof,to generate sequence reads obtained from each of the subsets ofantigen-specific T cells; (e) for each reaction mixture in the pluralityof reaction mixtures, identifying a plurality of antigen-specific TCRchains, or portion thereof, by comparing the sequence reads obtainedfrom each of the subsets of antigen-specific T cells in step (d) to thesequence reads obtained from unstimulated T cells in step (a), whereinthe frequency of the sequence reads for the WT-1 antigen-specific TCRchains, or portion thereof, is increased in the sequence reads obtainedfrom the subsets of antigen-specific T cells compared to the frequencyof sequence reads for the antigen-specific TCR chains, or portionthereof, in the sequence reads obtained from unstimulated T cells; and(f) identifying one or more TCR chains, or portion thereof, specific forone of the antigens from the one or more TCR chains, or a portionthereof, identified in step (e), wherein the frequency of the sequencereads for the one or more TCR chains, or portion thereof, specific forthe antigen is increased in the sequence reads obtained from each of thesubsets of antigen-specific T cells in which the antigen was present inthe corresponding reaction mixture.

In some embodiments, the present disclosure provides a recombinant Tcell receptor (TCR) that binds Wilms' tumor antigen-1 (WT1), comprisingan alpha chain and a beta chain, wherein the beta chain comprises a CDR3sequence comprising or consisting of an amino acid sequence set forth inSEQ ID NOs: 22 or 30. In some embodiments, the alpha chain comprises aCDR3 sequence comprising or consisting of an amino acid sequence setforth in SEQ ID NOs: 19 or 27. In some embodiments, the beta chaincomprises a CDR2 sequence comprising or consisting of an amino acidsequence set forth in SEQ ID NOs: 21 or 29. In some embodiments, thealpha chain comprises a CDR2 sequence comprising or consisting of anamino acid set forth in SEQ ID NOs: 18 or 26. In some embodiments, thebeta chain comprises a CDR1 sequence comprising or consisting of anamino acid sequence set forth in SEQ ID NOs: 20 or 28. In someembodiments, the alpha chain comprises a CDR1 sequence comprising orconsisting of an amino acid sequence set forth in SEQ ID NOs: 17 or 25.

In some embodiments, the present disclosure provides a recombinant TCRcomprising an alpha chain CDR1 comprising or consisting of an amino acidsequence according to SEQ ID NO: 17; an alpha chain CDR2 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 18; analpha chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 19; a beta chain CDR1 comprising or consistingof an amino acid sequence according to SEQ ID NO: 20; a beta chain CDR2comprising or consisting of an amino acid sequence according to SEQ IDNO: 21; and a beta chain CDR3 comprising or consisting of an amino acidsequence according to SEQ ID NO: 22.

In some embodiments, the present disclosure provides a recombinant TCRcomprising an alpha chain CDR1 comprising or consisting of an amino acidsequence according to SEQ ID NO: 25; an alpha chain CDR2 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 26; analpha chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 27; a beta chain CDR1 comprising or consistingof an amino acid sequence according to SEQ ID NO:28; a beta chain CDR2comprising or consisting of an amino acid sequence according to SEQ IDNO: 29; and a beta chain CDR3 comprising or consisting of an amino acidsequence according to SEQ ID NO: 30.

In some embodiments, the present disclosure provides a recombinant TCRthat binds WT1, comprising an alpha chain and a beta chain, wherein theamino acid sequence of the alpha chain TCR comprises an amino acidsequence having at least 95%, at least 96%, at least 97%, at least 98%,or at least 99% identity to a sequence set forth in SEQ ID NOs: 23 or 31and the beta chain comprises an amino acid sequence having at least 95%,at least 96%, at least 97%, at least 98%, or at least 99% identity to asequence set forth in SEQ ID NOs: 24 or 32. In some embodiments, theamino acid sequence of the alpha chain TCR comprises an amino acidsequence having at least 95%, at least 96%, at least 97%, at least 98%,or at least 99% identity to a sequence set forth in SEQ ID NO: 23 andthe beta chain comprises an amino acid sequence having at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identity to asequence set forth in SEQ ID NO: 24. In some embodiments, the amino acidsequence of the alpha chain TCR comprises an amino acid sequence havingat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to a sequence set forth in SEQ ID NO: 31 and the beta chaincomprises an amino acid sequence having at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity to a sequence setforth in SEQ ID NO:32. In some embodiments, the amino acid sequence ofthe alpha chain TCR comprises or consists of an amino acid sequence setforth in SEQ ID NO: 23 and the beta chain comprises or consists of anamino acid sequence set forth in SEQ ID NO: 24. In some embodiments, theamino acid sequence of the alpha chain TCR comprises or consists of anamino acid sequence set forth in SEQ ID NO: 31 and the beta chaincomprises or consists of an amino acid sequence set forth in SEQ IDNO:32.

In some embodiments, the present disclosure provides a recombinant TCRcomprising an alpha chain and a beta chain, wherein the beta chaincomprises a CDR3 sequence comprising an amino acid sequence having atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to an amino acid sequence set forth in SEQID NOs: 6, 14, 22, 30, 38, or 46. In some embodiments, the presentdisclosure provides a recombinant TCR that binds WT1, comprising analpha chain and a beta chain, wherein the alpha chain comprises a CDR3sequence comprising an amino acid sequence having at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to an amino acid sequence set forth in SEQ ID NOs: 3, 11, 19,27, 35, or 43. In some embodiments, the beta chain comprises a CDR2sequence comprising an amino acid sequence having at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to an amino acid sequence set forth in SEQ ID NOs: 5, 13, 21,29, 37, or 45. In some embodiments, the alpha chain comprises a CDR2sequence comprising an amino acid sequence having at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to an amino acid sequence set forth in SEQ ID NOs: 2, 10, 18,26, 34, or 42. In some embodiments, the beta chain comprises a CDR1sequence comprising an amino acid sequence having at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to an amino acid sequence set forth in SEQ ID NOs: 4, 12, 20,28, 36, or 44. In some embodiments, the alpha chain comprises a CDR1sequence comprising an amino acid sequence having at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to an amino acid sequence set forth in SEQ ID NOs: 1, 9, 17,25, 33, or 41.

In some embodiments, the present disclosure provides a recombinant TCR,wherein the TCR comprises a) an alpha chain CDR3 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 3 and abeta chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 6; or b) an alpha chain CDR3 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 11 and abeta chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 14; or c) an alpha chain CDR3 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 19 and abeta chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 22; or d) an alpha chain CDR3 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 27 and abeta chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 30; or e) an alpha chain CDR3 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 35 and abeta chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 38; or f) an alpha chain CDR3 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 43 and abeta chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 46.

In some embodiments, the present disclosure provides a recombinant TCRcomprising an alpha chain CDR1 comprising or consisting of an amino acidsequence according to SEQ ID NO: 1; an alpha chain CDR2 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 2; an alphachain CDR3 comprising or consisting of an amino acid sequence accordingto SEQ ID NO: 3; a beta chain CDR1 comprising or consisting of an aminoacid sequence according to SEQ ID NO: 4; a beta chain CDR2 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 5; and abeta chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 6.

In some embodiments, the present disclosure provides a recombinant TCRcomprising an alpha chain CDR1 comprising or consisting of an amino acidsequence according to SEQ ID NO: 9; an alpha chain CDR2 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 10; analpha chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 11; a beta chain CDR1 comprising or consistingof an amino acid sequence according to SEQ ID NO: 12; a beta chain CDR2comprising or consisting of an amino acid sequence according to SEQ IDNO: 13; and a beta chain CDR3 comprising or consisting of an amino acidsequence according to SEQ ID NO: 14.

In some embodiments, the present disclosure provides a recombinant TCRcomprising an alpha chain CDR1 comprising or consisting of an amino acidsequence according to SEQ ID NO: 17; an alpha chain CDR2 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 18; analpha chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 19; a beta chain CDR1 comprising or consistingof an amino acid sequence according to SEQ ID NO: 20; a beta chain CDR2comprising or consisting of an amino acid sequence according to SEQ IDNO: 21; and a beta chain CDR3 comprising or consisting of an amino acidsequence according to SEQ ID NO: 22.

In some embodiments, the present disclosure provides a recombinant TCRcomprising an alpha chain CDR1 comprising or consisting of an amino acidsequence according to SEQ ID NO: 25; an alpha chain CDR2 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 26; analpha chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 27; a beta chain CDR1 comprising or consistingof an amino acid sequence according to SEQ ID NO:28; a beta chain CDR2comprising or consisting of an amino acid sequence according to SEQ IDNO: 29; and a beta chain CDR3 comprising or consisting of an amino acidsequence according to SEQ ID NO: 30.

In some embodiments, the present disclosure provides a recombinant TCRcomprising an alpha chain CDR1 comprising or consisting of an amino acidsequence according to SEQ ID NO: 33; an alpha chain CDR2 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 34; analpha chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 35; a beta chain CDR1 comprising or consistingof an amino acid sequence according to SEQ ID NO: 36; a beta chain CDR2comprising or consisting of an amino acid sequence according to SEQ IDNO: 37; and a beta chain CDR3 comprising or consisting of an amino acidsequence according to SEQ ID NO: 38.

In some embodiments, the present disclosure provides a recombinant TCRcomprising an alpha chain CDR1 comprising or consisting of an amino acidsequence according to SEQ ID NO: 41; an alpha chain CDR2 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 42; analpha chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 43; a beta chain CDR1 comprising or consistingof an amino acid sequence according to SEQ ID NO: 44; a beta chain CDR2comprising or consisting of an amino acid sequence according to SEQ IDNO: 45; and a beta chain CDR3 comprising or consisting of an amino acidsequence according to SEQ ID NO: 46.

In some embodiments, the present disclosure provides a recombinant TCRcomprising an alpha chain and a beta chain, wherein the amino acidsequence of the alpha chain TCR comprises an amino acid sequence havingat least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identity to a sequence set forth in SEQ ID NOs: 7, 15, 23,31, 39, or 47 and the beta chain comprises an amino acid sequence havingat least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identity to a sequence set forth in SEQ ID NOs: 8, 16, 24,32, 40, or 48. In some embodiments, the amino acid sequence of the alphachain TCR comprises an amino acid sequence having at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identityto a sequence set forth in SEQ ID NO: 7 and the beta chain comprises anamino acid sequence having at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity to a sequence setforth in SEQ ID NO: 8. In some embodiments, the amino acid sequence ofthe alpha chain TCR comprises an amino acid sequence having at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identity to a sequence set forth in SEQ ID NO: 15 and the beta chaincomprises an amino acid sequence having at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identity to asequence set forth in SEQ ID NO: 16. In some embodiments, the amino acidsequence of the alpha chain TCR comprises an amino acid sequence havingat least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identity to a sequence set forth in SEQ ID NO: 23 and thebeta chain comprises an amino acid sequence having at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to a sequence set forth in SEQ ID NO: 24. In some embodiments,the amino acid sequence of the alpha chain TCR comprises an amino acidsequence having at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99% identity to a sequence set forth in SEQ IDNO: 31 and the beta chain comprises an amino acid sequence having atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity to a sequence set forth in SEQ ID NO: 32. In someembodiments, the amino acid sequence of the alpha chain TCR comprises anamino acid sequence having at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity to a sequence setforth in SEQ ID NO: 39 and the beta chain comprises an amino acidsequence having at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99% identity to a sequence set forth in SEQ IDNO: 40. In some embodiments, the amino acid sequence of the alpha chainTCR comprises an amino acid sequence having at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99% identity to asequence set forth in SEQ ID NO: 47 and the beta chain comprises anamino acid sequence having at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity to a sequence setforth in SEQ ID NO: 48.

In some embodiments, the present disclosure provides a recombinant TCRcomprising an alpha chain and a beta chain, wherein the amino acidsequence of the alpha chain TCR comprises or consists of an amino acidsequence selected from the group consisting of SEQ ID NOs: 7, 15, 23,31, 39, and 47 and the beta chain comprises or consists of an amino acidsequence selected from the group consisting of SEQ ID NOs: 8, 16, 24,32, 40, and 48. In some embodiments, the amino acid sequence of thealpha chain TCR comprises or consists of an amino acid sequence setforth in SEQ ID NO: 7 and the beta chain comprises or consists of anamino acid sequence set forth in SEQ ID NO: 8. In some embodiments, theamino acid sequence of the alpha chain TCR comprises or consists of anamino acid sequence set forth in SEQ ID NO: 15 and the beta chaincomprises or consists of an amino acid sequence set forth in SEQ ID NO:16. In some embodiments, the amino acid sequence of the alpha chain TCRcomprises or consists of an amino acid sequence set forth in SEQ ID NO:23 and the beta chain comprises or consists of an amino acid sequenceset forth in SEQ ID NO: 24. In some embodiments, the amino acid sequenceof the alpha chain TCR comprises or consists of an amino acid sequenceset forth in SEQ ID NO: 31 and the beta chain comprises or consists ofan amino acid sequence set forth in SEQ ID NO: 32. In some embodiments,the amino acid sequence of the alpha chain TCR comprises or consists ofan amino acid sequence set forth in SEQ ID NO: 39 and the beta chaincomprises or consists of an amino acid sequence set forth in SEQ ID NO:40. In some embodiments, the amino acid sequence of the alpha chain TCRcomprises or consists of an amino acid sequence set forth in SEQ ID NO:47 and the beta chain comprises or consists of an amino acid sequenceset forth in SEQ ID NO: 48.

In some embodiments, the present disclosure provides a recombinant TCRthat is capable of binding to an epitope on the WT1 protein epitope orcapable of eliciting a functional T cell response. In some embodiments,the T cell response is measured by CD69 expression or T cell cytolysis.In some embodiments, the epitope comprises a sequence according to SEQID NO: 97. In some embodiments, the TCR is capable of binding to aWT1/HLA-A2 complex with an interaction half-life (t_(1/2)) of less than100 seconds, or about 30 seconds to about 1000 seconds. In someembodiments, the TCR binds a WT1/HLA-A2 complex and is capable ofactivating a functional T cell response and having an EC₅₀ less thanabout 100 nM, less than about 10 nM, less than about 1 nM, less thanabout 500 pM, less than about 100 pM, or less than about 1 pM.

In some embodiments, the present disclosure provides a recombinant TCR,wherein the TCR is a soluble TCR. In some embodiments, the TCR iscoupled to an antibody or fragment thereof. In some embodiments, theantibody or fragment thereof is a monoclonal antibody, scFv, or Fab. Insome embodiments, the antibody or fragment thereof is an anti-CD3 scFvor an anti-CD3 Fab.

In some embodiments, the present disclosure provides a method fortreating cancer in a subject in need thereof, comprising administeringto the subject an effective amount of a composition comprising arecombinant TCR described herein. In some embodiments, the presentdisclosure provides a recombinant TCR for use in a method of treatingcancer in a subject in need thereof. In some embodiments, the cancer isa leukemia.

In some embodiments, the present disclosure provides a recombinant hostcell comprising an exogenous TCR, wherein the exogenous TCR comprises anexogenous alpha chain and an exogenous beta chain, wherein the alphachain and the beta chain each comprise CDR1, CDR2, and CDR3, wherein thealpha chain CDR3 comprises an amino acid sequence having at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 3, 11, 19, 27, 35, and 43. In someembodiments, the present disclosure provides a recombinant host cellcomprising an exogenous TCR, wherein the exogenous TCR comprises anexogenous alpha chain and an exogenous beta chain, wherein the alphachain and the beta chain each comprise CDR1, CDR2, and CDR3, wherein thebeta chain CDR3 comprises an amino acid sequence having at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 6, 14, 22, 30, 38, and 46. In someembodiments, the alpha chain comprises a CDR2 sequence comprising anamino acid sequence having at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 2, 10,18, 26, 34, and 42. In some embodiments, the beta chain comprises a CDR2sequence comprising an amino acid sequence having at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 5, 13, 21, 29, 37, and 45. In some embodiments, the alphachain comprises a CDR1 sequence comprising an amino acid sequence havingat least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% sequence identity to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 1, 9, 17, 25, 33, and 41. In someembodiments, the beta chain comprises a CDR1 sequence comprising anamino acid sequence having at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 4, 12,20, 28, 36, and 44.

In some embodiments, the present disclosure provides a recombinant hostcell comprising an exogenous TCR, wherein the exogenous TCR comprises a)an alpha chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 3 and a beta chain CDR3 comprising or consistingof an amino acid sequence according to SEQ ID NO: 6; or b) an alphachain CDR3 comprising or consisting of an amino acid sequence accordingto SEQ ID NO: 11 and a beta chain CDR3 comprising or consisting of anamino acid sequence according to SEQ ID NO: 14; or c) an alpha chainCDR3 comprising or consisting of an amino acid sequence according to SEQID NO: 19 and a beta chain CDR3 comprising or consisting of an aminoacid sequence according to SEQ ID NO: 22; or d) an alpha chain CDR3comprising or consisting of an amino acid sequence according to SEQ IDNO: 27 and a beta chain CDR3 comprising or consisting of an amino acidsequence according to SEQ ID NO: 30; or e) an alpha chain CDR3comprising or consisting of an amino acid sequence according to SEQ IDNO: 35 and a beta chain CDR3 comprising or consisting of an amino acidsequence according to SEQ ID NO: 38; or f) an alpha chain CDR3comprising or consisting of an amino acid sequence according to SEQ IDNO: 43 and a beta chain CDR3 comprising or consisting of an amino acidsequence according to SEQ ID NO: 46.

In some embodiments, the present disclosure provides a recombinant hostcell comprising an exogenous TCR, wherein the exogenous TCR comprises analpha chain CDR1 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 1; an alpha chain CDR2 comprising or consistingof an amino acid sequence according to SEQ ID NO: 2; an alpha chain CDR3comprising or consisting of an amino acid sequence according to SEQ IDNO: 3; a beta chain CDR1 comprising or consisting of an amino acidsequence according to SEQ ID NO: 4; a beta chain CDR2 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 5; and abeta chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 6.

In some embodiments, the present disclosure provides a recombinant hostcell comprising an exogenous TCR, wherein the exogenous TCR comprises analpha chain CDR1 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 9; an alpha chain CDR2 comprising or consistingof an amino acid sequence according to SEQ ID NO: 10; an alpha chainCDR3 comprising or consisting of an amino acid sequence according to SEQID NO: 11; a beta chain CDR1 comprising or consisting of an amino acidsequence according to SEQ ID NO: 12; a beta chain CDR2 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 13; and abeta chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 14.

In some embodiments, the present disclosure provides a recombinant hostcell comprising an exogenous TCR, wherein the exogenous TCR comprises analpha chain CDR1 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 17; an alpha chain CDR2 comprising or consistingof an amino acid sequence according to SEQ ID NO: 18; an alpha chainCDR3 comprising or consisting of an amino acid sequence according to SEQID NO: 19; a beta chain CDR1 comprising or consisting of an amino acidsequence according to SEQ ID NO: 20; a beta chain CDR2 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 21; and abeta chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 22.

In some embodiments, the present disclosure provides a recombinant hostcell comprising an exogenous TCR, wherein the exogenous TCR comprises analpha chain CDR1 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 25; an alpha chain CDR2 comprising or consistingof an amino acid sequence according to SEQ ID NO: 26; an alpha chainCDR3 comprising or consisting of an amino acid sequence according to SEQID NO: 27; a beta chain CDR1 comprising or consisting of an amino acidsequence according to SEQ ID NO: 28; a beta chain CDR2 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 29; and abeta chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 30.

In some embodiments, the present disclosure provides a recombinant hostcell comprising an exogenous TCR, wherein the exogenous TCR comprises analpha chain CDR1 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 33; an alpha chain CDR2 comprising or consistingof an amino acid sequence according to SEQ ID NO: 34; an alpha chainCDR3 comprising or consisting of an amino acid sequence according to SEQID NO: 35; a beta chain CDR1 comprising or consisting of an amino acidsequence according to SEQ ID NO: 36; a beta chain CDR2 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 37; and abeta chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 38.

In some embodiments, the present disclosure provides a recombinant hostcell comprising an exogenous TCR, wherein the exogenous TCR comprises analpha chain CDR1 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 41; an alpha chain CDR2 comprising or consistingof an amino acid sequence according to SEQ ID NO: 42; an alpha chainCDR3 comprising or consisting of an amino acid sequence according to SEQID NO: 43; a beta chain CDR1 comprising or consisting of an amino acidsequence according to SEQ ID NO: 44; a beta chain CDR2 comprising orconsisting of an amino acid sequence according to SEQ ID NO: 45; and abeta chain CDR3 comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 46.

In some embodiments, the present disclosure provides a recombinant hostcell comprising an exogenous TCR, wherein the amino acid sequence of theTCR alpha chain comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 7, 15, 23, 31, 39, and 47 and the TCR betachain comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 8, 16, 24, 32, 40, and 48. In someembodiments, the amino acid sequence of the alpha chain TCR comprises anamino acid sequence having at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity to SEQ ID NO: 7 andthe beta chain comprises an amino acid sequence having at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to SEQ ID NO: 8. In some embodiments, the amino acid sequenceof the alpha chain TCR comprises an amino acid sequence having at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identity to SEQ ID NO: 15 and the beta chain comprises an amino acidsequence having at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99% identity to SEQ ID NO: 16. In someembodiments, the amino acid sequence of the alpha chain TCR comprises anamino acid sequence having at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity to SEQ ID NO: 23 andthe beta chain comprises an amino acid sequence having at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to SEQ ID NO: 24.3

In some embodiments, the amino acid sequence of the alpha chain TCRcomprises an amino acid sequence having at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identity to SEQID NO: 31 and the beta chain comprises an amino acid sequence having atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity to SEQ ID NO: 32. In some embodiments, the amino acidsequence of the alpha chain TCR comprises an amino acid sequence havingat least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identity to SEQ ID NO: 39 and the beta chain comprises anamino acid sequence having at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity to SEQ ID NO: 40. Insome embodiments, the amino acid sequence of the alpha chain TCRcomprises an amino acid sequence having at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identity to SEQID NO: 47 and the beta chain comprises an amino acid sequence having atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity to SEQ ID NO: 48.

In some embodiments, the present disclosure provides a recombinant hostcell comprising an exogenous TCR wherein the amino acid sequence of thealpha chain TCR comprises or consists of an amino acid sequence setforth in SEQ ID NO: 7 and the beta chain comprises or consists of anamino acid sequence set forth in SEQ ID NO: 8. In some embodiments, thepresent disclosure provides a recombinant host cell comprising anexogenous TCR wherein the amino acid sequence of the alpha chain TCRcomprises or consists of an amino acid sequence set forth in SEQ ID NO:15 and the beta chain comprises or consists of an amino acid sequenceset forth in SEQ ID NO: 16. In some embodiments, the present disclosureprovides a recombinant host cell comprising an exogenous TCR wherein theamino acid sequence of the alpha chain TCR comprises or consists of anamino acid sequence set forth in SEQ ID NO: 23 and the beta chaincomprises or consists of an amino acid sequence set forth in SEQ ID NO:24. In some embodiments, the present disclosure provides a recombinanthost cell comprising an exogenous TCR wherein the amino acid sequence ofthe alpha chain TCR comprises or consists of an amino acid sequence setforth in SEQ ID NO: 31 and the beta chain comprises or consists of anamino acid sequence set forth in SEQ ID NO: 32. In some embodiments, thepresent disclosure provides a recombinant host cell comprising anexogenous TCR wherein the amino acid sequence of the alpha chain TCRcomprises or consists of an amino acid sequence set forth in SEQ ID NO:39 and the beta chain comprises or consists of an amino acid sequenceset forth in SEQ ID NO: 40. In some embodiments, the present disclosureprovides a recombinant host cell comprising an exogenous TCR wherein theamino acid sequence of the alpha chain TCR comprises or consists of anamino acid sequence set forth in SEQ ID NO: 47 and the beta chaincomprises or consists of an amino acid sequence set forth in SEQ ID NO:48.

In some embodiments, the present disclosure provides a recombinant hostcell comprising an exogenous TCR wherein the host cell is ahematopoietic progenitor cell. In some embodiments, the host cell is animmune cell. In some embodiments, the immune cell is a T cell, NK cell,or NK T cell. In some embodiments, the T cell is a naïve T cell, aneffector T cell, a central memory T cell, an effector memory T cell, aCD4+ T cell, a CD8+ T cell, an alpha/beta T cell, a gamma/delta T cell,or any combination thereof. In some embodiments, the T cell is a CD8+ Tcell.

In some embodiments, the present disclosure provides a host cellcomprising an exogenous TCR, wherein the exogenous TCR comprises one ormore modifications to reduce the frequency of mis-pairing betweenexogenous TCR alpha and beta chains with endogenous TCR alpha and betachains in the T cell. In some embodiments, the one or more modificationsare selected from the group consisting of one or more cysteinemutations, one or more knob-and-hole mutations, dimerization peptidesfused to the exogenous TCR alpha and/or beta chains, or one or morelinker molecules linking the exogenous TCR alpha and beta chains. Insome embodiments, the one or more cysteine mutations is present in theconstant region of the exogenous TCR.

In some embodiments, the present disclosure provides a host cellcomprising an exogenous TCR, wherein the cell is further engineered toexpress an exogenous immune activating molecule. In some embodiments,the immune activating molecule is a cytokine or a ligand. In someembodiments, the immune activating molecule is selected from the groupconsisting of IL-12, TNFα, IFNγ, IL2, IL-7, IL-15, IL-18, CD40L, andIL-21.

In some embodiments, the present disclosure provides a method fortreating cancer in a subject in need thereof, comprising administeringto the subject a recombinant host cell described herein. In someembodiments, the present disclosure provides use of a recombinant hostcell described herein in a method for treating cancer. In someembodiments, the cancer is a leukemia.

In some embodiments, the present disclosure provides a method fortreating cancer in a subject in need thereof, comprising the steps of:a) isolating T cells from peripheral blood mononuclear cells (PBMC)obtained from the subject; b) transducing or transfecting the T cellswith a nucleic acid comprising a sequence selected from the groupconsisting of SEQ ID NOs: 55, 56, 63, 64, 71, 72, 79, 80, 87, 88, 95,and/or 96; and c) administering the transduced or transfected T cells tothe subject. In some embodiments, the present disclosure provides amethod for treating cancer in a subject in need thereof, comprising thesteps of: a) obtaining T cells from a donor source; b) transducing ortransfecting the T cells with a nucleic acid comprising a sequenceselected from the group consisting of SEQ ID NOs: 55, 56, 63, 64, 71,72, 79, 80, 87, 88, 95, and/or 96; and c) administering the transducedor transfected T cells to the subject. In some embodiments, the T cellsare transduced or transfected a nucleic acid sequence comprising orconsisting of a) SEQ ID NOs: 55 and 56; or b) SEQ ID NOs: 63 and 64; orc) SEQ ID NOs: 71 and 72; or d) SEQ ID NOs: 79 and 80; or e) SEQ ID NOs:87 and 88; or f) SEQ ID NOs: 95 and 96.

In some embodiments, the nucleic acid sequence is comprised in a vector.In some embodiments, the T cells are further transduced or transfectedwith a nucleic acid encoding an immune activating molecule. In someembodiments, the immune activating molecule is a ligand or a cytokine.In some embodiments, the immune activating molecule is selected from thegroup consisting of IL-12, TNFα, IFNγ, IL2, IL-7, IL-15, IL-18, CD40L,and IL-21. In some embodiments, the method further comprises expandingthe transduced or transfected T cells prior to administration to thesubject.

In some embodiments, the present disclosure provides a method ofproducing a T cell comprising exogenous alpha and beta TCR chains, themethod comprising transducing or transfecting an isolated T cell withone or more polynucleotides, wherein the polynucleotides comprises asequence selected from the group consisting of SEQ ID NOs: 55, 56, 63,64, 71, 72, 79, 80, 87, 88, 95, and/or 96; and incubating the cell underconditions suitable for expression of the exogenous alpha and beta TCRchains by the cell. In some embodiments, the isolated T cell istransduced or transfected with polynucleotides comprising or consistingof a). SEQ ID NOs: 55 and 56; or b). SEQ ID NOs: 63 and 64; or c). SEQID NOs: 71 and 72 or d). SEQ ID NOs: 79 and 80 or e). SEQ ID NOs: 87 and88 or f). SEQ ID NOs: 95 and 96. In some embodiments, the method furthercomprises transducing or transfecting the isolated T cell with a nucleicacid encoding an immune activating molecule. In some embodiments, theimmune activating molecule is a ligand or a cytokine. In someembodiments, the immune activating molecule is selected from the groupconsisting of IL-12, TNFα, IFNγ, IL2, IL-7, IL-15, IL-18, CD40L, andIL-21.

In some embodiments, the present disclosure provides a pharmaceuticalcomposition comprising a recombinant TCR described herein. In someembodiments, the present disclosure provides a pharmaceuticalcomposition comprising a combination of TCRs comprising amino acidsequences selected from the group consisting of SEQ ID NOs: 1-48. Insome embodiments, the present disclosure provides a vector capable ofexpressing a nucleic acid molecule encoding a TCR alpha chain having anamino acid sequence selected from the group consisting of SEQ ID NOs: 7,15, 23, 31, 39, or 47. In some embodiments, the present disclosureprovides a vector capable of expressing a nucleic acid molecule encodinga TCR beta chain having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 8, 16, 24, 32, 40, or 48. In some embodiments,the present disclosure provides a cell comprising a vector describedherein.

In some embodiments, the present disclosure provides a cell comprising afirst vector capable of expressing a nucleic acid molecule encoding aTCR alpha chain comprising an amino acid sequence having at least 90%,95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 7, 15, 23, 31, 39, and47 and a second vector capable of expressing a nucleic acid moleculeencoding a TCR beta chain comprising an amino acid sequence having atleast 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 8, 16, 24,32, 40, and 48.

In some embodiments, the first vector is capable of expressing a nucleicacid molecule encoding a TCR alpha chain comprising or consisting of anamino acid sequence according to SEQ ID NO: 7 and the second vector iscapable of expressing a nucleic acid molecule encoding a TCR beta chaincomprising or consisting of an amino acid sequence according to SEQ IDNO: 8. In some embodiments, the first vector is capable of expressing anucleic acid molecule encoding a TCR alpha chain comprising orconsisting of an amino acid sequence according to SEQ ID NO: 15 and thesecond vector is capable of expressing a nucleic acid molecule encodinga TCR beta chain comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 16. In some embodiments, the first vector iscapable of expressing a nucleic acid molecule encoding a TCR alpha chaincomprising or consisting of an amino acid sequence according to SEQ IDNO: 23 and the second vector is capable of expressing a nucleic acidmolecule encoding a TCR beta chain comprising or consisting of an aminoacid sequence according to SEQ ID NO: 24. In some embodiments, the firstvector is capable of expressing a nucleic acid molecule encoding a TCRalpha chain comprising or consisting of an amino acid sequence accordingto SEQ ID NO: 31 and the second vector is capable of expressing anucleic acid molecule encoding a TCR beta chain comprising or consistingof an amino acid sequence according to SEQ ID NO: 32. In someembodiments, the first vector is capable of expressing a nucleic acidmolecule encoding a TCR alpha chain comprising or consisting of an aminoacid sequence according to SEQ ID NO: 39 and the second vector iscapable of expressing a nucleic acid molecule encoding a TCR beta chaincomprising or consisting of an amino acid sequence according to SEQ IDNO: 40. In some embodiments, the first vector is capable of expressing anucleic acid molecule encoding a TCR alpha chain comprising orconsisting of an amino acid sequence according to SEQ ID NO: 47 and thesecond vector is capable of expressing a nucleic acid molecule encodinga TCR beta chain comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 48.

In some embodiments, the present disclosure provides a cDNA encoding a Tcell receptor (TCR) that binds Wilms' tumor antigen-1 (WT1), comprisingan alpha chain and a beta chain, wherein the beta chain comprises a CDR3sequence comprising an amino acid sequence having at least 90%, 95%,96%, 97%, 98%, or 99% sequence identity to an amino acid sequence setforth in SEQ ID NOs: 6, 14, 22, 30, 38, or 46. In some embodiments, thepresent disclosure provides a cDNA encoding a TCR that binds WT1,comprising an alpha chain and a beta chain, wherein the alpha chaincomprises a CDR3 sequence comprising an amino acid sequence having atleast 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acidsequence set forth in SEQ ID NOs: 3, 11, 19, 27, 35, or 43.

In some embodiments, the present disclosure provides a T cell receptor(TCR) or a portion thereof that binds WT-1, comprising an alpha chainand a beta chain, wherein the beta chain comprises a CDR3 sequencecomprising an amino acid sequence having at least 90% sequence identityto an amino acid sequence set forth in SEQ ID NOs: 6, 14, 22, 30, 38, or46, wherein the antigen specificity of the TCR or portion thereof isdetermined by a method comprising the steps of: (a) sequencingrecombined nucleic acids encoding one or more TCR chain(s), or a portionthereof, from a first portion of a sample comprising T cells to generatea first multiplicity of sequence reads obtained from T cells prior toWT-1 antigen exposure of the sample, wherein the sequencing ishigh-throughput sequencing; (b) partitioning a second portion of thesample comprising T cells into a plurality of reaction mixtures andexposing each reaction mixture of the plurality of reaction mixtures toWT-1 antigens; (c) for each reaction mixture in the plurality ofreaction mixtures, separating T cells that interact with one or moreWT-1 antigens in the reaction mixture to obtain a subset of WT-1antigen-specific T cells, wherein each of the subsets ofantigen-specific T cells corresponds to one reaction mixture in theplurality of reaction mixtures; (d) for each of the subsets of WT-1antigen-specific T cells separated in step (c), sequencing recombinednucleic acids encoding one or more TCR chain(s), or a portion thereof,to generate a multiplicity of sequence reads obtained from each of thesubsets of antigen-specific T cells, wherein the sequencing ishigh-throughput sequencing; (e) for each reaction mixture in theplurality of reaction mixtures, identifying a plurality ofantigen-specific TCR chains, or portion thereof, by comparing themultiplicity of sequence reads obtained from each of the subsets of WT-1antigen-specific T cells in step (d) to the first multiplicity ofsequence reads obtained from unstimulated T cells in step (a), whereinthe frequency of the sequence reads for the WT-1 antigen-specific TCRchains, or portion thereof, is increased in the multiplicity of sequencereads obtained from the subsets of antigen-specific T cells compared tothe frequency of sequence reads for the antigen-specific TCR chains, orportion thereof, in the first multiplicity of sequence reads obtainedfrom unstimulated T cells; and (f) identifying one or more TCR chains,or portion thereof, specific for the WT-1 antigen from the one or moreTCR chains, or a portion thereof, identified in step (e), wherein thefrequency of the sequence reads for the one or more TCR chains, orportion thereof, specific for the antigen is increased in themultiplicity of sequence reads obtained from each of the subsets ofantigen-specific T cells in which the antigen was present in thecorresponding reaction mixture.

In some embodiments, the present disclosure provides a T cell receptor(TCR) or a portion thereof that binds WT-1, comprising an alpha chainand a beta chain, wherein the alpha chain comprises a CDR3 sequencecomprising an amino acid sequence having at least 90% sequence identityto an amino acid sequence set forth in SEQ ID NOs: 3, 11, 19, 27, 35, or43, wherein the antigen specificity of the TCR or portion thereof isdetermined by a method comprising the steps of: (a) sequencingrecombined nucleic acids encoding one or more TCR chain(s), or a portionthereof, from a first portion of a sample comprising T cells to generatea first multiplicity of sequence reads obtained from T cells prior toWT-1 antigen exposure of the sample, wherein the sequencing ishigh-throughput sequencing; (b) partitioning a second portion of thesample comprising T cells into a plurality of reaction mixtures andexposing each reaction mixture of the plurality of reaction mixtures toWT-1 antigens; (c) for each reaction mixture in the plurality ofreaction mixtures, separating T cells that interact with one or moreWT-1 antigens in the reaction mixture to obtain a subset of WT-1antigen-specific T cells, wherein each of the subsets ofantigen-specific T cells corresponds to one reaction mixture in theplurality of reaction mixtures; (d) for each of the subsets of WT-1antigen-specific T cells separated in step (c), sequencing recombinednucleic acids encoding one or more TCR chain(s), or a portion thereof,to generate a multiplicity of sequence reads obtained from each of thesubsets of antigen-specific T cells, wherein the sequencing ishigh-throughput sequencing; (e) for each reaction mixture in theplurality of reaction mixtures, identifying a plurality ofantigen-specific TCR chains, or portion thereof, by comparing themultiplicity of sequence reads obtained from each of the subsets of WT-1antigen-specific T cells in step (d) to the first multiplicity ofsequence reads obtained from unstimulated T cells in step (a), whereinthe frequency of the sequence reads for the WT-1 antigen-specific TCRchains, or portion thereof, is increased in the multiplicity of sequencereads obtained from the subsets of antigen-specific T cells compared tothe frequency of sequence reads for the antigen-specific TCR chains, orportion thereof, in the first multiplicity of sequence reads obtainedfrom unstimulated T cells; and (f) identifying one or more TCR chains,or portion thereof, specific for the WT-1 antigen from the one or moreTCR chains, or a portion thereof, identified in step (e), wherein thefrequency of the sequence reads for the one or more TCR chains, orportion thereof, specific for the antigen is increased in themultiplicity of sequence reads obtained from each of the subsets ofantigen-specific T cells in which the antigen was present in thecorresponding reaction mixture.

In some embodiments, the present disclosure provides a T cell receptor(TCR) or a portion thereof that binds WT-1, comprising an alpha chainand a beta chain, wherein the beta chain comprises a CDR3 sequencecomprising an amino acid sequence having at least 90% sequence identityto an amino acid sequence set forth in SEQ ID NOs: 6, 14, 22, 30, 38, or46, wherein the antigen specificity of the TCR or portion thereof isdetermined by a method comprising the steps of: dividing a tissue sampleinto a first subset and a second subset; sequencing recombined nucleicacids encoding a TCR or a portion thereof from the first subset toprovide sequence reads from which clonotypes are determined; exposingthe second subset to WT-1 antigen; separating T cells from the secondsubset that interact with the antigen to obtain an enriched T cellsample; sequencing recombined nucleic acids encoding a TCR or a portionthereof from the enriched T cell sample to provide sequence reads fromwhich clonotypes are determined; and identifying WT-1 antigen-specific Tcells in the tissue sample as T cells whose clonotype frequenciesincrease in the enriched T cell sample relative to the frequencies ofthe same clonotype in the first subset.

In some embodiments, the present disclosure provides a T cell receptor(TCR) or a portion thereof that binds WT-1, comprising an alpha chainand a beta chain, wherein the alpha chain comprises a CDR3 sequencecomprising an amino acid sequence having at least 90% sequence identityto an amino acid sequence set forth in SEQ ID NOs: 3, 11, 19, 27, 35, or43, wherein the antigen specificity of the TCR or portion thereof isdetermined by a method comprising the steps of: dividing a tissue sampleinto a first subset and a second subset; sequencing recombined nucleicacids encoding a TCR or a portion thereof from the first subset toprovide sequence reads from which clonotypes are determined; exposingthe second subset to WT-1 antigen; separating T cells from the secondsubset that interact with the antigen to obtain an enriched T cellsample; sequencing recombined nucleic acids encoding a TCR or a portionthereof from the enriched T cell sample to provide sequence reads fromwhich clonotypes are determined; and identifying WT-1 antigen-specific Tcells in the tissue sample as T cells whose clonotype frequenciesincrease in the enriched T cell sample relative to the frequencies ofthe same clonotype in the first subset.

In some embodiments, the beta chain comprises a CDR2 sequence comprisingan amino acid sequence having at least 90% sequence identity to an aminoacid sequence set forth in SEQ ID NOs: 5, 13, 21, 29, 37, or 45. In someembodiments, the alpha chain comprises a CDR2 sequence comprising anamino acid sequence having at least 90% sequence identity to an aminoacid sequence set forth in SEQ ID NOs: 2, 10, 18, 26, 34, or 42. In someembodiments, the beta chain comprises a CDR1 sequence comprising anamino acid sequence having at least 90% sequence identity to an aminoacid sequence set forth in SEQ ID NO: 4, 12, 20, 28, 36, or 44. In someembodiments, the alpha chain comprises a CDR1 sequence comprising anamino acid sequence having at least 90% sequence identity to an aminoacid sequence set forth in SEQ ID NO: 1, 9, 17, 25, 33, or 41. In someembodiments, the TCR is capable of binding to an epitope on the WT1protein epitope or capable of eliciting a functional T cell response.

These above-characterized aspects, as well as other aspects, of thepresent invention are exemplified in a number of illustratedimplementations and applications, some of which are shown in the figuresand characterized in the claims section that follows. However, the abovesummary is not intended to describe each illustrated embodiment or everyimplementation of the invention.

BRIEF DESCRIPTION OF THE FIGURES

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention is obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A and FIG. 1B show the avidity and half-life of WT1 tetramers witheither the gene expression constructs for either eJH30_WT1C_5 (FIG. 1A)or eJH30_WT1C_8 (FIG. 1B) were electroporated into TCR-deficient cells.WT1-expressing T cells were then stained with WT1/HLA-A2-PE tetramers.Tetramer dissociation over time was plotted as the natural logarithm oftetramer fluorescence intensity (minus background) over time. The slopeof this line provided the negative of the dissociation rate (−kd), and(ln(2))/kd) represents the half-life (t_(1/2)) of WT1/HLA-A2 tetramer'sinteraction with TCR on the cells.

FIG. 2 shows the functional avidity of the TCRs provided herein. TCRswere expressed in a TCR deficient cell line, and incubated with cellsthat had been pulsed with the indicated concentration (nM) of WT1peptide (SEQ ID NO: 97). Mean fluorescence intensity (MFI) of CD69 onCD3+ cells was assessed by FACS after a 4 hour incubation to assess therelative potency of the TCRs.

FIG. 3 is a graph showing the antigen-specific killing capacity of cellsexpressing the TCRs provided herein. The TCRs were expressed in CD8+ Tcells expanded from healthy donors. The CD8+ T cells (effector cells; E)were then incubated with a 50:50 mixture of cells that had been pulsedwith 10 μg/mL WT1 peptide (SEQ ID NO: 97) and cells pulsed with a dye(target cells; T), at various E:T ratios. The percent of live targetcells following the incubation period was assessed by FACS and plottedagainst the E:T ratio to show the antigen-specific killing capacity ofthe cells expressing the recombinant TCRs.

FIG. 4 is a graph showing the antigen-specific killing capacity of cellsexpressing the TCRs provided herein. The TCRs were expressed in CD8+ Tcells expanded from healthy donors. The CD8+ T cells (effector cells; E)were then incubated with a 50:50 mixture of cells that had been pulsedwith the indicated amount of the WT1 peptide (SEQ ID NO: 97) and cellspulsed with a dye (target cells; T), at a ratio of 2:1 E:T. The percentof live target cells following the incubation period was assessed byFACS and plotted against peptide concentration to show theantigen-specific killing potency of the two recombinant TCRs.

FIG. 5A-FIG. 5F are graphs showing the antigen specific killing capacityof cells expressing eJH30_7 (FIG. 5A), eJH30_5 (FIG. 5B), eJH30_8 (FIG.5C), eJH52_13 (FIG. 5D), eJH64_6 (FIG. 5E), and eJH64_9 (FIG. 5F).Endogenous, presentation-deficient T2 cells were incubated with theindicated concentrations of WT1 peptide, then incubated 16 hours withpolyclonal T cells expressing either no exogenous TCR (‘polyclonal Tcells’), or expressing the indicated WT1-specific TCR. The remainingviable peptide-loaded T2 cells were enumerated by FACS (in comparison toan internal control population of labeled non-WT1-presenting T2 cells)and expressed as % specific lysis.

FIG. 6A-FIG. 6F are graphs showing IFNγ secretion by cells expressingeJH30_7 (FIG. 6A), eJH30_5 (FIG. 6B), eJH30_8 (FIG. 6C), eJH52_13 (FIG.6D), eJH64_6 (FIG. 6E), and eJH64_9 (FIG. 6F). Endogenous,presentation-deficient T2 cells were incubated with the indicatedconcentrations of WT1 peptide, then incubated 16 hours with polyclonal Tcells expressing either no exogenous TCR (‘polyclonal T cells’), orexpressing the indicated WT1-specific TCR. T cells' IFNγ secretions wereassessed after 16 hours.

FIG. 7 is a graph showing the capacity of cells expressing eJH52_13 toinduce specific lysis of cells expressing endogenous WT1 antigen and/orexogenous WT1 peptide.

FIG. 8 is a graph showing the capacity of cells expressing the TCRindicated on the x-axis to induce specific lysis of cells expressingendogenous WT1 antigen and/or exogenous WT1 peptide.

FIG. 9 shows IFNγ secretion from eJH52_13 expressing T cells in thepresence of endogenous WT1 expression only, exogenous WT1 peptide, orWT1-epitope-containing ivtRNA.

FIGS. 10A-10D show the functional avidity of eJH30_7 (30_7; FIG. 10A),eJH52_13 (52_13; FIG. 10B), eJH64_6 (64_6; FIG. 10C), and eJH64_9 (64_9;FIG. 10D). TCRs were expressed in a TCR deficient cell line, andincubated with cells that had been pulsed with the indicatedconcentration (nM) of WT1 peptide (SEQ ID NO: 97). Mean fluorescenceintensity (MFI) of CD69 on CD3+ cells was assessed by FACS after a 2hour incubation to assess the relative potency of the TCRs.

FIG. 11 shows the relative functional avidity of each of eJH30_7,eJH52_13, eJH64_6, and eJH64_9. The results shown in FIGS. 10A-10D areprovided in a single graph in FIG. 11 .

FIG. 12A illustrates diagrammatically steps of one embodiment of theinvention for matching TCR alpha and TCR beta chains from separatelysequenced molecules.

FIG. 12B illustrates diagrammatically steps of another embodiment of theinvention for determining either TCR alpha or TCR beta chains thatoriginate from the same T cell or heavy and light chain immunoglobulinsthat originate from the same B cell.

FIG. 12C illustrates diagrammatically an embodiment of the invention foridentifying antigen-specific T cells that interact with a singleantigen.

FIG. 12D illustrates diagrammatically an embodiment of the invention foridentifying antigen-specific T cells that interact with a plurality ofantigens.

FIG. 12E illustrates steps of an embodiment of the invention forphysical identification of antigen-specific T cells using single cellmethodology.

FIG. 12F illustrates a PCA scheme for linking target sequences wherepairs of internal primers have complementary.

FIG. 13A-FIG. 13C show a two-staged PCR scheme for amplifying TCR betagenes.

FIG. 14A-FIG. 14B illustrate details of embodiments for determining anucleotide sequence of the PCR product of FIG. 13C.

FIG. 15 illustrates an example of a tissue sample divided or aliquotedinto a plurality of subsets 1 through 10 and examples of differentsubpluralities of subsets of the plurality.

FIG. 16A-FIG. 16B illustrate an embodiment of the invention fordetermining T-cells and TCRs specific for a plurality of antigens.

FIG. 17A-FIG. 17B show data for identification of CMV pp65₄₉₅-specific Tcell clonotypes from sorted pentamer+ T cells. FIG. 17A shows clonotypefrequencies from CMV pp65₄₉₅ pentamer+ versus pentamer-CD8+ T cells froma characterized CMV responder. The 8 dots (enclosed in dashed ellipse600) indicate clonotypes greater than 10-fold enriched and exceeding a20-cell equivalent minimum frequency threshold in the sorted (pentamer+)population. FIG. 17B shows that all 8 clonotypes identified in FIG. 17Aare enriched in (unsorted) PBMCs from the same individual. The dotsenclosed by the dashed ellipse (602) indicate clonotypes identified inFIG. 17A.

FIG. 18A-FIG. 18C show data for identification of CMV pp65₄₉₅-specific Tcell clonotypes from sorted responding cells following peptideincubation. Clonotype frequencies from sorted responding CD37+ cellsfollowing CMV pp65₄₉₅ peptide incubation versus either sortednon-responding CD137− cells (FIG. 18A) or unsorted PBMCs (FIG. 18B). The9 data points enclosed by dashed ellipse (700) in panel A indicateclonotypes greater than 10-fold enriched and exceeding a 20-cellequivalent minimum frequency threshold in the sorted (CD137+)population. Data points enclosed in dashed ellipse (702) in panel Bindicate those clonotypes identified in panel A. Clonotypes identifiedin panel A are not enriched in sorted CD137+ cells versus CD137− T cells(FIG. 18C arrows indicating data points corresponding to those enclosedby ellipses in FIG. 18A and FIG. 18B) following incubation withoutpeptide.

FIG. 19A-FIG. 19B illustrate the overlap between clonotypes identifiedin pentamer-based and CD137-based assays. In FIG. 19A, the plot showsclonotype frequencies of the 8 clonotypes (enclosed by dashed ellipse800) identified in the pentamer analyses in the clonotype profiles ofCD137+ responding cells following CMV pp65₄₉₅ peptide incubation versussorted non-responding CD137− cells. In FIG. 19B, the plot showsclonotype frequencies of the 9 clonotypes (enclosed by dashed ellipse802) identified in the CD137 assay analyses in the clonotype profiles ofsorted CMV pp65₄₉₅ pentamer+ cells versus pentamer-cells. 8/9 of theseclonotypes are overlapping with those identified in FIG. 19A.

FIG. 20A-FIG. 20D shows data for identification of low-frequency CMVpp65₄₉₅-specific T cell clonotypes following peptide incubation andproliferation. In FIG. 20A, clonotype frequencies from sortedproliferating CD8+ T cells following CMV pp65₄₉₅ peptide incubation atday 6 versus fresh unsorted PBMCs. The 16 data points (enclosed bydashed ellipse 900) indicate clonotypes greater than 10-fold enrichedand exceeding 1/10,000 minimum frequency threshold in the sortedproliferating cells. FIG. 20B shows data of clonotype frequencies fromsorted proliferating CD8+ T cells following incubation without peptideat day 6 versus fresh unsorted PBMCs. Clonotypes (represented by datapoints enclosed by dashed ellipses (902) are those identified in FIG.20A. FIG. 20C shows data of clonotype frequencies from CMV pp65₄₉₅pentamer+ versus pentamer− CD8+ T cells. Dashed ellipses (904) and arrow(906) indicate the 16 clonotypes identified in the proliferation assaywhose results are represented in FIG. 20A with those clonotypesidentified in the CMV pp65₄₉₅ pentamer+ versus pentamer− CD8+ T cellcomparison. FIG. 20D shows data of clonotype frequencies from CMVpp65₄₉₅ pentamer+ versus pentamer− CDK+ cells. Data points enclosed bydashed ellipses (908) and designated by arrows (910) indicate the 25clonotypes identified in a variant of the proliferation assay describedabove with those clonotypes identified in the CMV pp65₄₉₅ pentamer+versus pentamer− CD8-4 T cell comparison. In this assay a pool of 138overlapping peptides from pp65 was used instead of the single pp65₄₉₅peptide.

FIG. 21A-FIG. 21E illustrate embodiments of coalescing sequence reads.FIG. 21A illustrates concepts of clonotypes in sequence space anddistances between closely related clonotypes. FIG. 21B is a flow chartillustrating one embodiment of a method for distinguishing genuinelydifferent clonotypes from clonotypes that differ solely by sequencingerrors (which should be coalesced). FIG. 21C illustrates the form of anumerical function used in one embodiment for determining whether or notto coalesce related clonotypes. FIG. 21D and FIG. 21E illustrate the useof sequence trees in a method of coalescing sequence reads.

DETAILED DESCRIPTION

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of molecularbiology, bioinformatics, cell biology, and biochemistry, which arewithin the skill of the art. Such conventional techniques include, butare not limited to, sampling and analysis of blood cells, nucleic acidsequencing and analysis, and the like. Specific illustrations ofsuitable techniques can be had by reference to the example herein below.However, other equivalent conventional procedures can, of course, alsobe used. Such conventional techniques and descriptions can be found instandard laboratory manuals.

I. Definitions

Unless otherwise specifically defined herein, terms and symbols ofnucleic acid chemistry, biochemistry, genetics, and molecular biologyused herein follow those of standard treatises and texts in the field,e.g. Kornberg and Baker, DNA Replication, Second Edition (W. H. Freeman,New York, 1992); Lehninger, Biochemistry, Second Edition (WorthPublishers, New York, 1975); Strachan and Read, Human MolecularGenetics, Second Edition (Wiley-Liss, New York, 1999); Abbas et al,Cellular and Molecular Immunology, 6^(th) edition (Saunders, 2007).

“Activation” or “immune activation” or “activated”, especially inreference to T-cells, means a phase of an adaptive immune response thatfollows the antigen recognition phase (during which antigen-specificlymphocytes bind to antigens) and is characterized by proliferation oflymphocytes and their differentiation into effector cells, e.g. Abbas etal, Cellular and Molecular Immunology, Fourth Edition, (W. B. SaundersCompany, 2000). Activation of T cells may be associated with secretionof certain cytokines that are detectable using conventional assays, suchas an ELISPOT assay, and may be associated with the expression ofcharacteristic cell surface markers, such as CD25, CD134, CD69, CD137,CD154, or the like, e.g. Gratama et al, Cytometry A, 73A: 971-974(2008).

“Aligning” means a method of comparing a test sequence, such as asequence read, to one or more reference sequences to determine whichreference sequence or which portion of a reference sequence is closestbased on some sequence distance measure. An exemplary method of aligningnucleotide sequences is the Smith Waterman algorithm. Distance measuresmay include Hamming distance, Levenshtein distance, or the like.Distance measures may include a component related to the quality valuesof nucleotides of the sequences being compared.

“Amplicon” means the product of a polynucleotide amplification reaction;that is, a clonal population of polynucleotides, which may be singlestranded or double stranded, which are replicated from one or morestarting sequences. The one or more starting sequences may be one ormore copies of the same sequence, or they may be a mixture of differentsequences. Preferably, amplicons are formed by the amplification of asingle starting sequence. Amplicons may be produced by a variety ofamplification reactions whose products comprise replicates of the one ormore starting, or target, nucleic acids. In one aspect, amplificationreactions producing amplicons are “template-driven” in that base pairingof reactants, either nucleotides or oligonucleotides, have complementsin a template polynucleotide that are required for the creation ofreaction products. In one aspect, template-driven reactions are primerextensions with a nucleic acid polymerase or oligonucleotide ligationswith a nucleic acid ligase. Such reactions include, but are not limitedto, polymerase chain reactions (PCRs), linear polymerase reactions,nucleic acid sequence-based amplification (NASBAs), rolling circleamplifications, and the like, disclosed in the following references thatare incorporated herein by reference: Mullis et al, U.S. Pat. Nos.4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et al, U.S.Pat. No. 5,210,015 (real-time PCR with “taqman” probes); Wittwer et al,U.S. Pat. No. 6,174,670; Kacian et al, U.S. Pat. No. 5,399,491(“NASBA”); Lizardi, U.S. Pat. No. 5,854,033; Aono et al, Japanese patentpubl. JP 4-262799 (rolling circle amplification); and the like. In oneaspect, amplicons of the invention are produced by PCRs. Anamplification reaction may be a “real-time” amplification if a detectionchemistry is available that permits a reaction product to be measured asthe amplification reaction progresses, e.g. “real-time PCR” describedbelow, or “real-time NASBA” as described in Leone et al, Nucleic AcidsResearch, 26: 2150-2155 (1998), and like references. As used herein, theterm “amplifying” means performing an amplification reaction. A“reaction mixture” means a solution containing all the necessaryreactants for performing a reaction, which may include, but not belimited to, buffering agents to maintain pH at a selected level during areaction, salts, co-factors, scavengers, and the like.

“Clonotype” means a recombined nucleotide sequence of a lymphocyte whichencodes an immune receptor or a portion thereof. More particularly,clonotype means a recombined nucleotide sequence of a T cell or B cellwhich encodes a T cell receptor (TCR) or B cell receptor (BCR), or aportion thereof. In various embodiments, clonotypes may encode all or aportion of a VDJ rearrangement of IgH, a DJ rearrangement of IgH, a VJrearrangement of IgK, a VJ rearrangement of IgL, a VDJ rearrangement ofTCR beta, a DJ rearrangement of TCR beta, a VJ rearrangement of TCRalpha, a VJ rearrangement of TCR gamma, a VDJ rearrangement of TCRdelta, a VD rearrangement of TCR delta, a Kde-V rearrangement, or thelike. Clonotypes may also encode translocation breakpoint regionsinvolving immune receptor genes, such as Bc11-IgH or Bc11-IgH. In oneaspect, clonotypes have sequences that are sufficiently long torepresent or reflect the diversity of the immune molecules that they arederived from; consequently, clonotypes may vary widely in length. Insome embodiments, clonotypes have lengths in the range of from 25 to 400nucleotides; in other embodiments, clonotypes have lengths in the rangeof from 25 to 200 nucleotides.

“Clonotype profile” means a listing of distinct clonotypes and theirrelative abundances that are derived from a population of lymphocytes.Typically, the population of lymphocytes are obtained from a tissuesample. The term “clonotype profile” is related to, but more generalthan, the immunology concept of immune “repertoire” as described inreferences, such as the following: Arstila et al, Science, 286: 958-961(1999); Yassai et al, Immunogenetics, 61: 493-502 (2009); Kedzierska etal, Mol. Immunol., 45(3): 607-618 (2008); and the like. The term“clonotype profile” includes a wide variety of lists and abundances ofrearranged immune receptor-encoding nucleic acids, which may be derivedfrom selected subsets of lymphocytes (e.g. tissue-infiltratinglymphocytes, immunophenotypic subsets, or the like), or which may encodeportions of immune receptors that have reduced diversity as compared tofull immune receptors. In some embodiments, clonotype profiles maycomprise at least 10³ distinct clonotypes; in other embodiments,clonotype profiles may comprise at least 10⁴ distinct clonotypes; inother embodiments, clonotype profiles may comprise at least 10⁵ distinctclonotypes; in other embodiments, clonotype profiles may comprise atleast 10⁶ distinct clonotypes. In such embodiments, such clonotypeprofiles may further comprise abundances or relative frequencies of eachof the distinct clonotypes.

In one aspect, a clonotype profile is a set of distinct recombinednucleotide sequences (with their abundances) that encode T cellreceptors (TCRs) or B cell receptors (BCRs), or fragments thereof,respectively, in a population of lymphocytes of an individual, whereinthe nucleotide sequences of the set have a one-to-one correspondencewith distinct lymphocytes or their clonal subpopulations forsubstantially all of the lymphocytes of the population. In one aspect,nucleic acid segments defining clonotypes are selected so that theirdiversity (i.e. the number of distinct nucleic acid sequences in theset) is large enough so that substantially every T cell or B cell orclone thereof in an individual carries a unique nucleic acid sequence ofsuch repertoire. That is, preferably each different clone of a samplehas different clonotype. In other aspects of the invention, thepopulation of lymphocytes corresponding to a repertoire may becirculating B cells, or may be circulating T cells, or may besubpopulations of either of the foregoing populations, including but notlimited to, CD4+ T cells, or CD8+ T cells, or other subpopulationsdefined by cell surface markers, or the like. Such subpopulations may beacquired by taking samples from particular tissues, e.g. bone marrow, orlymph nodes, or the like, or by sorting or enriching cells from a sample(such as peripheral blood) based on one or more cell surface markers,size, morphology, or the like. In still other aspects, the population oflymphocytes corresponding to a repertoire may be derived from diseasetissues, such as a tumor tissue, an infected tissue, or the like. In oneembodiment, a clonotype profile comprising human TCR beta chains orfragments thereof comprises a number of distinct nucleotide sequences inthe range of from 0.1×10⁶ to 1.8×10⁶, or in the range of from 0.5×10⁶ to1.5×10⁶, or in the range of from 0.8×10⁶ to 1.2×10⁶. In anotherembodiment, a clonotype profile comprising human IgH chains or fragmentsthereof comprises a number of distinct nucleotide sequences in the rangeof from 0.1×10⁶ to 1.8×10⁶, or in the range of from 0.5×10⁶ to 1.5×10⁶,or in the range of from 0.8×10⁶ to 1.2×10⁶.

In a particular embodiment, a clonotype profile of the inventioncomprises a set of nucleotide sequences encoding substantially allsegments of the V(D)J region of an IgH chain. In one aspect,“substantially all” as used herein means every segment having a relativeabundance of 0.001 percent or higher; or in another aspect,“substantially all” as used herein means every segment having a relativeabundance of 0.0001 percent or higher. In another particular embodiment,a clonotype profile of the invention comprises a set of nucleotidesequences that encodes substantially all segments of the V(D)J region ofa TCR beta chain. In another embodiment, a clonotype profile of theinvention comprises a set of nucleotide sequences having lengths in therange of from 25-200 nucleotides and including segments of the V, D, andJ regions of a TCR beta chain. In another embodiment, a clonotypeprofile of the invention comprises a set of nucleotide sequences havinglengths in the range of from 25-200 nucleotides and including segmentsof the V, D, and J regions of an IgH chain. In another embodiment, aclonotype profile of the invention comprises a number of distinctnucleotide sequences that is substantially equivalent to the number oflymphocytes expressing a distinct IgH chain. In another embodiment, aclonotype profile of the invention comprises a number of distinctnucleotide sequences that is substantially equivalent to the number oflymphocytes expressing a distinct TCR beta chain. In still anotherembodiment, “substantially equivalent” means that with ninety-ninepercent probability a clonotype profile will include a nucleotidesequence encoding an IgH or TCR beta or portion thereof carried orexpressed by every lymphocyte of a population of an individual at afrequency of 0.001 percent or greater. In still another embodiment,“substantially equivalent” means that with ninety-nine percentprobability a repertoire of nucleotide sequences will include anucleotide sequence encoding an IgH or TCR beta or portion thereofcarried or expressed by every lymphocyte present at a frequency of0.0001 percent or greater. In some embodiments, clonotype profiles arederived from samples comprising from 10³ to 10⁷ lymphocytes. Suchnumbers of lymphocytes may be obtained from peripheral blood samples offrom 1-10 mL.

“Coalescing” means treating two candidate clonotypes with sequencedifferences as the same by determining that such differences are due toexperimental or measurement error and not due to genuine biologicaldifferences. In one aspect, a sequence of a higher frequency candidateclonotype is compared to that of a lower frequency candidate clonotypeand if predetermined criteria are satisfied then the number of lowerfrequency candidate clonotypes is added to that of the higher frequencycandidate clonotype and the lower frequency candidate clonotype isthereafter disregarded. That is, the read counts associated with thelower frequency candidate clonotype are added to those of the higherfrequency candidate clonotype.

“Complementarity determining regions” (CDRs) mean regions of animmunoglobulin (i.e., antibody) or T cell receptor where the moleculecomplements an antigen's conformation, thereby determining themolecule's specificity and contact with a specific antigen. T cellreceptors and immunoglobulins each have three CDRs: CDR1 and CDR2 arefound in the variable (V) domain, and CDR3 includes some of V, all ofdiverse (D) (heavy chains only) and joint (J), and some of the constant(C) domains.

“Data structure” means an organization of information, usually in acomputer or memory device, for better algorithm efficiency. Exemplarydata structures include queues, stacks, linked lists, heaps, hashtables, arrays, trees, and the like. Data structures may havesubstructures that correspond to units of information or to subsets ofrelated information. For example, arrays have rows and columns ofentries; trees have nodes, branches, subtrees, and leaves; or the like.In one aspect, a data structure used herein is a sequence tree, an arrayor a hash table.

As used herein, the term “exogenous” refers to a molecule, nucleic acid,protein, or structure that is introduced into the cell by genetic orbiochemical means. By contrast, an “endogenous” molecule, nucleic acid,protein, or structure is one that is present in the particular celland/or in the particular cell at its developmental stage. An exogenousmolecule, nucleic acid, protein, or structure can be the same type as anendogenous molecule, nucleic acid, protein, or structure found withinthe cell, or may be a type of molecule, nucleic acid, protein, orstructure that is not normally found in the cell.

The term “exogenous TCR,” as used herein, refers to a recombinant TCRexpressed in a cell via introduction of exogenous coding sequences for aTCR. Thus, the cell comprising an exogenous TCR is capable of expressinga TCR that is not natively expressed in that cell.

The term “isolated,” as used herein, refers to a biological componentsuch as a nucleic acid, peptide, protein, or cell that has beensubstantially separated, produced apart from, or purified away fromother biological components of the organism in which the componentnaturally occurs. Nucleic acids, peptides, proteins, and cells that havebeen isolated thus include nucleic acids, peptides proteins, and cellsthat are purified by standard purification methods, or that are preparedby expression, for example expression in a host cell, or that arechemically synthesized. In some embodiments, the isolated cell is anautologous cell, meaning that it may is derived from the subject thatwill receive the resultant transduced or transformed cell. For example,in some embodiments, the isolated cells are derived from the PBMC and/orhematopoietic stem cells of the subject being treated.

“Microfluidics device” means an integrated system of one or morechambers, ports, and channels that are interconnected and in fluidcommunication and designed for carrying out an analytical reaction orprocess, either alone or in cooperation with an appliance or instrumentthat provides support functions, such as sample introduction, fluidand/or reagent driving means, temperature control, detection systems,data collection and/or integration systems, and the like. Microfluidicsdevices may further include valves, pumps, and specialized functionalcoatings on interior walls, e.g. to prevent adsorption of samplecomponents or reactants, facilitate reagent movement by electroosmosis,or the like. Such devices are usually fabricated in or as a solidsubstrate, which may be glass, plastic, or other solid polymericmaterials, and typically have a planar format for ease of detecting andmonitoring sample and reagent movement, especially via optical orelectrochemical methods. Features of a microfluidic device usually havecross-sectional dimensions of less than a few hundred square micrometersand passages typically have capillary dimensions, e.g. having maximalcross-sectional dimensions of from about 500 μm to about 0.1 μm.Microfluidics devices typically have volume capacities in the range offrom 1 μL to a few nL, e.g. 10-100 nL. The fabrication and operation ofmicrofluidics devices are well-known in the art as exemplified by thefollowing references that are incorporated by reference: Ramsey, U.S.Pat. Nos. 6,001,229; 5,858,195; 6,010,607; and U.S. Pat. No. 6,033,546;Soane et al, U.S. Pat. Nos. 5,126,022 and 6,054,034; Nelson et al, U.S.Pat. No. 6,613,525; Maher et al, U.S. Pat. No. 6,399,952; Ricco et al,International patent publication WO 02/24322; Bjornson et al,International patent publication WO 99/19717; Wilding et al, U.S. Pat.Nos. 5,587,128; 5,498,392; Sia et al, Electrophoresis, 24: 3563-3576(2003).

“Percent homologous,” “percent identical,” or like terms used inreference to the comparison of a reference sequence and another sequence(“comparison sequence”) mean that in an optimal alignment between thetwo sequences, the comparison sequence is identical to the referencesequence in a number of subunit positions equivalent to the indicatedpercentage, the subunits being nucleotides for polynucleotidecomparisons or amino acids for polypeptide comparisons. As used herein,an “optimal alignment” of sequences being compared is one that maximizesmatches between subunits and minimizes the number of gaps employed inconstructing an alignment. Percent identities may be determined withcommercially available implementations of algorithms, such as thatdescribed by Needleman and Wunsch, J. Mol. Biol., 48: 443-453(1970)(“GAP” program of Wisconsin Sequence Analysis Package, GeneticsComputer Group, Madison, Wis.), or the like. Other software packages inthe art for constructing alignments and calculating percentage identityor other measures of similarity include the “BestFit” program, based onthe algorithm of Smith and Waterman, Advances in Applied Mathematics, 2:482-489 (1981) (Wisconsin Sequence Analysis Package, Genetics ComputerGroup, Madison, Wis.). In other words, for example, to obtain apolynucleotide having a nucleotide sequence at least 95 percentidentical to a reference nucleotide sequence, up to five percent of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to five percent of thetotal number of nucleotides in the reference sequence may be insertedinto the reference sequence.

“Polymerase chain reaction,” or “PCR,” means a reaction for the in vitroamplification of specific DNA sequences by the simultaneous primerextension of complementary strands of DNA. In other words, PCR is areaction for making multiple copies or replicates of a target nucleicacid flanked by primer binding sites, such reaction comprising one ormore repetitions of the following steps: (i) denaturing the targetnucleic acid, (ii) annealing primers to the primer binding sites, and(iii) extending the primers by a nucleic acid polymerase in the presenceof nucleoside triphosphates. Usually, the reaction is cycled throughdifferent temperatures optimized for each step in a thermal cyclerinstrument. Particular temperatures, durations at each step, and ratesof change between steps depend on many factors well-known to those ofordinary skill in the art, e.g. exemplified by the references: McPhersonet al, editors, PCR: A Practical Approach and PCR2: A Practical Approach(IRL Press, Oxford, 1991 and 1995, respectively). For example, in aconventional PCR using Taq DNA polymerase, a double stranded targetnucleic acid may be denatured at a temperature >90° C., primers annealedat a temperature in the range 50-75° C., and primers extended at atemperature in the range 72-78° C.

The term “PCR” encompasses derivative forms of the reaction, includingbut not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR,multiplexed PCR, and the like. Reaction volumes range from a few hundrednanoliters, e.g. 200 nL, to a few hundred μL, e.g. 200 μL.

“Reverse transcription PCR,” or “RT-PCR,” means a PCR that is precededby a reverse transcription reaction that converts a target RNA to acomplementary single stranded DNA, which is then amplified, e.g. Tecottet al, U.S. Pat. No. 5,168,038, which patent is incorporated herein byreference.

“Real-time PCR” means a PCR for which the amount of reaction product,i.e. amplicon, is monitored as the reaction proceeds. There are manyforms of real-time PCR that differ mainly in the detection chemistriesused for monitoring the reaction product, e.g. Gelfand et al, U.S. Pat.No. 5,210,015 (“tagman”); Wittwer et al, U.S. Pat. Nos. 6,174,670 and6,569,627 (intercalating dyes); Tyagi et al, U.S. Pat. No. 5,925,517(molecular beacons); which patents are incorporated herein by reference.Detection chemistries for real-time PCR are reviewed in Mackay et al,Nucleic Acids Research, 30: 1292-1305 (2002), which is also incorporatedherein by reference.

“Nested PCR” means a two-stage PCR wherein the amplicon of a first PCRbecomes the sample for a second PCR using a new set of primers, at leastone of which binds to an interior location of the first amplicon. Asused herein, “initial primers” in reference to a nested amplificationreaction mean the primers used to generate a first amplicon, and“secondary primers” mean the one or more primers used to generate asecond, or nested, amplicon. “Multiplexed PCR” means a PCR whereinmultiple target sequences (or a single target sequence and one or morereference sequences) are simultaneously carried out in the same reactionmixture, e.g. Bernard et al, Anal. Biochem., 273: 221-228 (1999)(two-color real-time PCR). Usually, distinct sets of primers areemployed for each sequence being amplified. Typically, the number oftarget sequences in a multiplex PCR is in the range of from 2 to 50, orfrom 2 to 40, or from 2 to 30.

“Quantitative PCR” means a PCR designed to measure the abundance of oneor more specific target sequences in a sample or specimen. QuantitativePCR includes both absolute quantitation and relative quantitation ofsuch target sequences. Quantitative measurements are made using one ormore reference sequences or internal standards that may be assayedseparately or together with a target sequence. The reference sequencemay be endogenous or exogenous to a sample or specimen, and in thelatter case, may comprise one or more competitor templates. Typicalendogenous reference sequences include segments of transcripts of thefollowing genes: beta-actin, GAPDH, beta2-microglobulin, ribosomal RNA,and the like. Techniques for quantitative PCR are well-known to those ofordinary skill in the art, as exemplified in the following referencesthat are incorporated by reference: Freeman et al, Biotechniques, 26:112-126 (1999); Becker-Andre et al, Nucleic Acids Research, 17:9437-9447 (1989); Zimmerman et al, Biotechniques, 21: 268-279 (1996);Diviacco et al, Gene, 122: 3013-3020 (1992); Becker-Andre et al, NucleicAcids Research, 17: 9437-9446 (1989); and the like.

“Polymerase cycling assembly” or “PCA” reaction (also referred to hereinas “linked PCR”) means a PCR that comprises at least one pair of outerprimers and at least one pair of inner primers. An inner primer has a 3′portion that is complementary to a target nucleic acid (or itscomplement) and a 5′ portion that is complementary to the 5′ portion ofanother inner primer corresponding to a different target nucleic acid.

“Primer” means an oligonucleotide, either natural or synthetic that iscapable, upon forming a duplex with a polynucleotide template, of actingas a point of initiation of nucleic acid synthesis and being extendedfrom its 3′ end along the template so that an extended duplex is formed.Extension of a primer is usually carried out with a nucleic acidpolymerase, such as a DNA or RNA polymerase. The sequence of nucleotidesadded in the extension process is determined by the sequence of thetemplate polynucleotide. Usually primers are extended by a DNApolymerase. Primers usually have a length in the range of from 14 to 40nucleotides, or in the range of from 18 to 36 nucleotides. Primers areemployed in a variety of nucleic amplification reactions, for example,linear amplification reactions using a single primer, or polymerasechain reactions, employing two or more primers. Guidance for selectingthe lengths and sequences of primers for particular applications is wellknown to those of ordinary skill in the art, as evidenced by thefollowing references that are incorporated by reference: Dieffenbach,editor, PCR Primer: A Laboratory Manual, 2nd Edition (Cold Spring HarborPress, New York, 2003).

“Quality score” means a measure of the probability that a baseassignment at a particular sequence location is correct. A varietymethods are well known to those of ordinary skill for calculatingquality scores for particular circumstances, such as, for bases calledas a result of different sequencing chemistries, detection systems,base-calling algorithms, and so on. Generally, quality score values aremonotonically related to probabilities of correct base calling. Forexample, a quality score, or Q, of 10 may mean that there is a 90percent chance that a base is called correctly, a Q of 20 may mean thatthere is a 99 percent chance that a base is called correctly, and so on.For some sequencing platforms, particularly those usingsequencing-by-synthesis chemistries, average quality scores decrease asa function of sequence read length, so that quality scores at thebeginning of a sequence read are higher than those at the end of asequence read, such declines being due to phenomena such as incompleteextensions, carry forward extensions, loss of template, loss ofpolymerase, capping failures, deprotection failures, and the like.

“Sequence read” means a sequence of nucleotides determined from asequence or stream of data generated by a sequencing technique, whichdetermination is made, for example, by means of base-calling softwareassociated with the technique, e.g. base-calling software from acommercial provider of a DNA sequencing platform. A sequence readusually includes quality scores for each nucleotide in the sequence.Typically, sequence reads are made by extending a primer along atemplate nucleic acid, e.g. with a DNA polymerase or a DNA ligase. Datais generated by recording signals, such as optical, chemical (e.g. pHchange), or electrical signals, associated with such extension. Suchinitial data is converted into a sequence read.

“Sequence tag” (or “tag”) or “barcode” means an oligonucleotide that isattached, usually via a covalent bond, to another molecule or molecularcomplex and that is used to identify and/or track the other molecule ina reaction or a series of reactions. Sequence tags may vary widely insize and compositions; the following references, which are incorporatedherein by reference, provide guidance for selecting sets of sequencetags appropriate for particular embodiments: Brenner, U.S. Pat. No.5,635,400; Brenner and Macevicz, U.S. Pat. No. 7,537,897; Brenner et al,Proc. Natl. Acad. Sci., 97: 1665-1670 (2000); Church et al, Europeanpatent publication 0 303 459; Shoemaker et al, Nature Genetics, 14:450-456 (1996); Morris et al, European patent publication 0799897A1;Wallace, U.S. Pat. No. 5,981,179; and the like. Lengths and compositionsof sequence tags can vary widely, and the selection of particularlengths and/or compositions depends on several factors including,without limitation, how tags are used to generate a readout, e.g. via ahybridization reaction or via an enzymatic reaction, such asamplification and sequencing; whether they are labeled, e.g. with afluorescent dye or the like; the number of distinguishable sequence tagsrequired to unambiguously identify a set of molecules of interest, andthe like, and how different must tags of a set be in order to ensurereliable identification, e.g. freedom from cross hybridization,misidentification from sequencing errors, or the like. In someembodiments, sequence tags may each have a length within a range of from6 to 36 nucleotides, or from 4 to 30 nucleotides, or from 8 to 40nucleotides, or from 6 to 50 nucleotides, respectively; provided,however, that the term “sequence tag” may also be used in reference to asequence tag of the foregoing lengths sandwiched between a pair ofprimers that may be used to amplify or otherwise manipulate the sequencetag, for example, in order to identify it by DNA sequencing. In oneaspect, sets of sequence tags are used wherein each sequence tag of aset has a unique nucleotide sequence that differs from that of everyother tag of the same set by a plurality of bases; in some embodiments,such plurality is at least three bases; in another aspect, sets ofsequence tags are used wherein the sequence of each tag of a set differsfront that of every other tag of the same set by at least four bases.

“Sequence tree” means a tree data structure for representing nucleotidesequences. In one aspect, a tree data structure of the invention is arooted directed tree comprising nodes and edges that do not includecycles, or cyclical pathways. Edges from nodes of tree data structuresof the invention are usually ordered. Nodes and/or edges are structuresthat may contain, or be associated with, a value. Each node in a treehas zero or more child nodes, which by convention are shown below it inthe tree. A node that has a child is called the child's parent node. Anode has at most one parent. Nodes that do not have any children arecalled leaf nodes. The topmost node in a tree is called the root node.Being the topmost node, the root node will not have parents. It is thenode at which operations on the tree commonly begin (although somealgorithms begin with the leaf nodes and work up ending at the root).All other nodes can be reached from it by following edges or links.

The term “subject” is herein defined as vertebrate, particularly mammal,more particularly human. For purposes of research, the subject mayparticularly be at least one animal model, e.g., a mouse, rat and thelike.

The term “therapeutically effective amount,” as used herein, refers toan amount that elicits an immune-mediated therapeutic effect in thesubject. A therapeutic effect may include treatment of symptoms of adisease or disorder, or treatment of the underlying condition, and/orprophylaxis against development or worsening of a disease or disorder.In some embodiments, a “therapeutic vaccine” or “method of vaccination”and the like refers to a composition or method for eliciting an immuneresponse against a pathogen or a component of a pathogen, such as toproduce protective immunity (i.e., immunity that prevents or reducesseverity of the disease associated with the pathogen).

II. Identification of T-Cell Receptor Chains

In some embodiments, the present disclosure provides methods formatching pairs of immune receptor chains from populations of theirencoding nucleic acids that have been sequenced. In some embodiments,the methods described herein provide methods for the identification ofimmune receptor chains that are specific for a particular antigen andmethods of matching those antigen-specific immune receptor chains toform a complete immune receptor molecule (e.g., a complete TCR molecule,also referred to herein as a “reconstituted TCR”).

A. Antigens

An antigen may be any compound or composition capable of eliciting acell-mediated immune response (that is, an adaptive immune response),particularly in a mammal, such as a human. In some embodiments, anantigen may be any compound that can be recognized by a T cell in thecontext of the MHC molecule. More particularly, antigens include, but isnot limited to, cells, tissue extracts, tissue or cell lysates,proteins, individually or as a mixture, a plurality of proteins,peptides, mixtures of peptides, lipids, carbohydrates, sugars, and thelike. An antigen can be characteristic of a disease, such as aninfectious disease, an autoimmune disease, or a cancer. The antigen canbe, for example, a viral antigen, a bacterial antigen, a cancer antigen,etc. In some embodiments, an antigen is a cancer antigen or a viralantigen. By “cancer antigen” is meant any molecule (e.g., protein,peptide, lipid, carbohydrate, etc.) solely or predominantly expressed orover-expressed by a tumor cell or cancer cell, such that the antigen isassociated with the tumor or cancer.

A cancer antigen may be a cancer antigen of only one type of cancer ortumor, such that the cancer antigen is associated with or characteristicof only one type of cancer or tumor. Alternatively, a cancer antigen maybe a cancer antigen (e.g., may be characteristic) of more than one typeof cancer or tumor. For example, a cancer antigen may be expressed byboth breast and prostate cancer cells and not expressed at all bynormal, non-tumor, or non-cancer cells, or expressed only minimally. Acancer antigen may a melanoma cancer antigen, a breast cancer antigen ora molecule such as Wilms' tumor (WT)-1 which is expressed in a varietyof cancers, including chronic myelocytic leukemia, multiple myeloma,acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML),myelodysplastic syndrome, mesothelioma, ovarian cancer, breast cancer,prostate cancer, gastrointestinal cancers, lung cancer, colon cancer,thyroid cancer, head and neck cancer, glioblastoma, and sarcoma. Otherexemplary cancer antigens include those of the group consisting ofgp100, MART-1, NY-ESO-1, a member of the MAGE family of proteins, e.g.,MAGE-A1, mesothelin, Tyrosinase, TRP-1, TRP-2, PMSA, Her-2, and p53.

An antigen may be a viral antigen. In some embodiments, “viral antigen”means those antigens encoded by a part of a viral genome which can bedetected by a specific immunological response. Viral antigens include,for example, a viral coat protein, an influenza viral antigen, an HIVantigen, a Hepatitis B antigen, or a Hepatitis C antigen.

An antigen can be naturally, artificially, or synthetically produced.Thus, an antigen can be a synthetic, isolated, and/or purified protein,polypeptide, or peptide. Methods of making or obtaining such antigensare known in the art. For example, suitable methods of de novosynthesizing polypeptides and proteins (e.g., antigenic polypeptides andproteins) are described in Chan et al., Fmoc Solid Phase PeptideSynthesis, Oxford University Press, Oxford, United Kingdom, 2005;Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc.,2000; Epitope Mapping, ed. Westwoood et al., Oxford University Press,Oxford, United Kingdom, 2000; and U.S. Pat. No. 5,449,752. Also,polypeptides and proteins (e.g., antigenic polypeptides and proteins)can be produced using nucleic acids which encode the polypeptide orprotein using standard methods. See, for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, N Y, 1994. The nucleotide sequences of many antigens areknown in the art and are available from the GenBank database of theNational Center for Biotechnology Information (NCBI) website. Further,an antigen can be isolated and/or purified from a source, such as aplant, a bacterium, an insect, a mammal, e.g., a rat, a human, etc.Methods of isolation and purification are well-known in the art.

An antigen can be a free antigen, e.g., unbound antigenic peptide (e.g.,a free peptide), or can be a bound antigen, e.g., an MHC-peptidetetramer or an antigenic peptide presented by a carrier cell which waspulsed with the peptide.

In some embodiments, peripheral blood mononuclear cells (PBMCs) (forexample, which may be obtained from blood, for example, as aleukapheresis product) from a subject may be cultured directly in thepresence of antigen, to load antigen presenting cells (APCs) among thePBMCs with the antigen and to activate/stimulate antigen-specific Tcells present in the PBMC. In this regard, PBMC may be collected from anindividual, contacted with an antigen of interest, such as a tumorantigen, or a viral lysate, etc. In this manner, the APCs present in thePBMCs are loaded with the antigen, which is then presented to the Tcells present in the sample. In some embodiments, antigen-specific Tcells may be activated with peptide-MHC tetramers, see for exampleAltman, et al., Science 1998 Jun. 19; 280(5371):1821. In someembodiments, a protein antigen may be exposed to T cells indirectly bygenerating a set of peptides for binding to MHC molecules, where thesequences of the peptides are based on the amino acid sequence of theprotein, e.g. Stickler et al, Toxicol. Sci., 77(2): 280-289 (2004). Insome such embodiments, peptides are overlapping peptides covering theprotein. In some embodiments, peptides each have a size of from 10 to 20amino acids.

T cells can be obtained from a number of sources, including peripheralblood mononuclear cells, bone marrow, thymus, tissue biopsy, tumor,lymph node tissue, gut associated lymphoid tissue, mucosa associatedlymphoid tissue, spleen tissue, or any other lymphoid tissue, andtumors. T cells can be obtained from T cell lines and from autologous orallogeneic sources. T cells may be obtained from a single individual ora population of individuals, for example, a population of individual whoall suffer from the same disease, such as, a cancer or an infectiousdisease.

In some embodiments, cells from the circulating blood of an individualare obtained by apheresis or leukapheresis. The apheresis producttypically contains lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and platelets. In one embodiment, the cells collected byapheresis or leukapheresis may be washed to remove the plasma fractionand to place the cells in an appropriate buffer or media for subsequentprocessing steps. In one embodiment of the invention, the cells arewashed with phosphate buffered saline (PBS). In an alternativeembodiment, the wash solution lacks calcium and may lack magnesium ormay lack many if not all divalent cations. As those of ordinary skill inthe art would readily appreciate a washing step may be accomplished bymethods known to those in the art, such as by using a semi-automated“flow-through” centrifuge (for example, the Cobe 2991 cell processor,Baxter) according to the manufacturer's instructions. After washing, thecells may be re-suspended in a variety of biocompatible buffers, suchas, for example, Ca++/Mg++ free PBS. Alternatively, the undesirablecomponents of the apheresis sample may be removed and the cells directlyre-suspended in culture media.

In other embodiments, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and by centrifugation througha PERCOLL™ gradient. A specific subpopulation of T cells, such as CD28+,CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated bypositive or negative selection techniques. For example, CD3+, CD28+ Tcells can be positively selected using CD3/CD28 conjugated magneticbeads (e.g., DYNABEADS© M-450 CD3/CD28 T Cell Expander). In someembodiments, enrichment of a T cell population by negative selection canbe accomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One such method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4+ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8.

Another method for preparing T cells for stimulation is to freeze thecells after the washing step, which does not require themonocyte-removal step. Wishing not to be bound by theory, the freeze andsubsequent thaw step provides a more uniform product by removinggranulocytes and, to some extent, monocytes in the cell population.After the washing step that removes plasma and platelets, the cells maybe suspended in a freezing solution. While many freezing solutions andparameters are known in the art and will be useful in this context, onemethod involves using PBS containing 20% DMSO and 8% human serum albumin(HSA), or other suitable cell freezing media. This is then diluted 1:1with media so that the final concentration of DMSO and HSA are 10% and4%, respectively. The cells are then frozen to −80° C. at a rate of 10per minute and stored in the vapor phase of a liquid nitrogen storagetank.

B. Samples

Samples, or tissue samples, of T-cells (T lymphocytes) may include, forexample, helper T cells (effector T cells or Th cells), cytotoxic Tcells (CTLs), memory T cells, and regulatory T cells, as well as othercell types normally found in a tissue sample. In one aspect, a sample ofT cells includes at least 1,000 T cells; but more typically, a sampleincludes at least 10,000 T cells, and more typically, at least 100,000 Tcells. In another aspect, a sample includes a number of T cells in therange of 1000 to 100,000 cells.

Samples used in the methods of the invention can come from a variety oftissues as noted above, including, for example, tumor tissue, blood andblood plasma, lymph fluid, cerebrospinal fluid surrounding the brain andthe spinal cord, synovial fluid surrounding bone joints, and the like.In one embodiment, the sample is a blood sample. The blood sample can beabout 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, or 5.0 mL. The sample can be a tumor biopsy. Thebiopsy can be from, for example, from a tumor of the brain, liver, lung,heart, colon, kidney, or bone marrow. Any biopsy technique used by thoseskilled in the art can be used for isolating a sample from a subject.For example, a biopsy can be an open biopsy, in which general anesthesiais used. The biopsy can be a closed biopsy, in which a smaller cut ismade than in an open biopsy. The biopsy can be a core or incisionalbiopsy, in which part of the tissue is removed. The biopsy can be anexcisional biopsy, in which attempts to remove an entire lesion aremade. The biopsy can be a fine needle aspiration biopsy, in which asample of tissue or fluid is removed with a needle.

The sample can be a biopsy, e.g., a skin biopsy. The biopsy can be from,for example, brain, liver, lung, heart, colon, kidney, or bone marrow.Any biopsy technique used by those skilled in the art can be used forisolating a sample from a subject. For example, a biopsy can be an openbiopsy, in which general anesthesia is used. The biopsy can be a closedbiopsy, in which a smaller cut is made than in an open biopsy. Thebiopsy can be a core or incisional biopsy, in which part of the tissueis removed. The biopsy can be an excisional biopsy, in which attempts toremove an entire lesion are made. The biopsy can be a fine needleaspiration biopsy, in which a sample of tissue or fluid is removed witha needle.

As discussed more fully below, in some embodiments, a sample oflymphocytes is sufficiently large so that substantially every T cell orB cell with a distinct clonotype is represented therein, thereby forminga repertoire (as the term is used herein). In some embodiments, a sampleis taken that contains with a probability of ninety-nine percent everyclonotype of a population present at a frequency of 0.001 percent orgreater. In another embodiment, a sample is taken that contains with aprobability of ninety-nine percent every clonotype of a populationpresent at a frequency of 0.0001 percent or greater. In one embodiment,a sample of T cells includes at least a half million cells, and inanother embodiment such sample includes at least one million cells.

Blood samples are of particular interest and may be obtained usingconventional techniques, e.g. Innis et al, editors, PCR Protocols(Academic Press, 1990); or the like. For example, white blood cells maybe separated from blood samples using convention techniques, e.g.RosetteSep kit (Stem Cell Technologies, Vancouver, Canada). Bloodsamples may range in volume from 100 μL to 10 mL; in one aspect, bloodsample volumes are in the range of from 200 μL to 2 mL. Optionally,subsets of white blood cells, e.g. lymphocytes, may be further isolatedusing conventional techniques, e.g. fluorescently activated cell sorting(FACS)(Becton Dickinson, San Jose, Calif.), magnetically activated cellsorting (MACS)(Miltenyi Biotec, Auburn, Calif.), or the like.

Since the identifying recombinations are present in the DNA of eachindividual's adaptive immunity cells as well as their associated RNAtranscripts, either RNA or DNA can be sequenced in the methods of theprovided invention. A recombined sequence from a T-cell encoding a Tcell receptor molecule, or a portion thereof, is referred to as aclonotype. The DNA or RNA can correspond to sequences from T-cellreceptor (TCR) genes. For example, the DNA and RNA can correspond tosequences encoding alpha, beta, gamma, or delta chains of a TCR. In amajority of T-cells, the TCR is a heterodimer consisting of analpha-chain and beta-chain. The TCR alpha chain is generated by VJrecombination, and the beta chain receptor is generated by V(D)Jrecombination. For the TCR beta chain, in humans there are 48 Vsegments, 2 D segments, and 13 J segments. Several bases may be deletedand others added (called N and P nucleotides) at each of the twojunctions. In a minority of T-cells, the TCRs consist of gamma and deltachains. The TCR gamma chain is generated by VJ recombination, and theTCR delta chain is generated by V(D)J recombination (Kenneth Murphy,Paul Travers, and Mark Walport, Janeway's Immunology 7th edition,Garland Science, 2007).

C. Amplification of Nucleic Acid Populations

Amplicons of target populations of nucleic acids may be generated by avariety of amplification techniques. In one aspect of the invention,multiplex PCR is used to amplify members of a mixture of nucleic acids,particularly mixtures comprising recombined immune molecules such as Tcell receptors, or portions thereof. Guidance for carrying out multiplexPCRs of such immune molecules is found in the following references,which are incorporated by reference U.S. Pat. Nos. 8,236,503; 8,628,927;5,296,351; 5,837,447; 6,087,096; U.S. Patent Application Publication No.2006/0234234; European Patent Publication EP 1544308B1; and the like.

After amplification of DNA from the genome (or amplification of nucleicacid in the form of cDNA by reverse transcribing RNA), the individualnucleic acid molecules can be isolated, optionally re-amplified, andthen sequenced individually. Exemplary amplification protocols may befound in van Dongen et al, Leukemia, 17: 2257-2317 (2003) or van Dongenet al, U.S. Patent Application Publication No. 2006/0234234, which isincorporated by reference. Briefly, an exemplary protocol is as follows:Reaction buffer: ABI Buffer II or ABI Gold Buffer (Life Technologies,San Diego, Calif.); 50 μL final reaction volume; 100 ng sample DNA; 10pmol of each primer (subject to adjustments to balance amplification asdescribed below); dNTPs at 200 μM final concentration; MgCl₂ at 1.5 mMfinal concentration (subject to optimization depending on targetsequences and polymerase); Taq polymerase (1-2 U/tube); cyclingconditions: pre-activation 7 min at 95° C.; annealing at 60° C.; cyclingtimes: 30 s denaturation; 30 s annealing; 30 s extension. Polymerasesthat can be used for amplification in the methods of the invention arecommercially available and include, for example, Taq polymerase,AccuPrime polymerase, or Pfu. The choice of polymerase to use can bebased on whether fidelity or efficiency is preferred.

Real time PCR, picogreen staining, nanofluidic electrophoresis (e.g.LabChip) or UV absorption measurements can be used in an initial step tojudge the functional amount of amplifiable material.

In one aspect, multiplex amplifications are carried out so that relativeamounts of sequences in a starting population are substantially the sameas those in the amplified population, or amplicon. That is, multiplexamplifications are carried out with minimal amplification bias amongmember sequences of a sample population. In one embodiment, suchrelative amounts are substantially the same if each relative amount inan amplicon is within five fold of its value in the starting sample. Inanother embodiment, such relative amounts are substantially the same ifeach relative amount in an amplicon is within two fold of its value inthe starting sample. As discussed more fully below, amplification biasin PCR may be detected and corrected using conventional techniques sothat a set of PCR primers may be selected for a predetermined repertoirethat provide unbiased amplification of any sample.

In one embodiment, amplification bias may be avoided by carrying out atwo-stage amplification (as described in Faham and Willis, cited above)wherein a small number of amplification cycles are implemented in afirst, or primary, stage using primers having tails non-complementarywith the target sequences. The tails include primer binding sites thatare added to the ends of the sequences of the primary amplicon so thatsuch sites are used in a second stage amplification using only a singleforward primer and a single reverse primer, thereby eliminating aprimary cause of amplification bias. Preferably, the primary PCR willhave a small enough number of cycles (e.g. 5-10) to minimize thedifferential amplification by the different primers. The secondaryamplification is done with one pair of primers and hence the issue ofdifferential amplification is minimal. One percent of the primary PCR istaken directly to the secondary PCR. Thirty-five cycles (equivalent to−28 cycles without the 100 fold dilution step) used between the twoamplifications were sufficient to show a robust amplificationirrespective of whether the breakdown of cycles were: one cycle primaryand 34 secondary or 25 primary and 10 secondary. Even though ideallydoing only 1 cycle in the primary PCR may decrease the amplificationbias, there are other considerations. One aspect of this isrepresentation. This plays a role when the starting input amount is notin excess to the number of reads ultimately obtained. For example, if1,000,000 reads are obtained and starting with 1,000,000 input moleculesthen taking only representation from 100,000 molecules to the secondaryamplification would degrade the precision of estimating the relativeabundance of the different species in the original sample. The 100 folddilution between the 2 steps means that the representation is reducedunless the primary PCR amplification generated significantly more than100 molecules. This indicates that a minimum 8 cycles (256 fold), butmore comfortably 10 cycle (−1,000 fold), may be used. The alternative tothat is to take more than 1% of the primary PCR into the secondary butbecause of the high concentration of primer used in the primary PCR, abig dilution factor is can be used to ensure these primers do notinterfere in the amplification and worsen the amplification bias betweensequences. Another alternative is to add a purification or enzymaticstep to eliminate the primers from the primary PCR to allow a smallerdilution of it. In this example, the primary PCR was 10 cycles and thesecond 25 cycles.

D. Generating Sequence Reads for Clonotypes

Any high-throughput technique for sequencing nucleic acids can be usedin the method of the invention. Preferably, such technique has acapability of generating in a cost-effective manner a volume of sequencedata from which at least 1000 clonotypes can be determined, andpreferably, from which at least 10,000 to 1,000,000 clonotypes can bedetermined. DNA sequencing techniques include classic dideoxy sequencingreactions (Sanger method) using labeled terminators or primers and gelseparation in slab or capillary, sequencing by synthesis usingreversibly terminated labeled nucleotides, pyrosequencing, 454sequencing, allele specific hybridization to a library of labeledoligonucleotide probes, sequencing by synthesis using allele specifichybridization to a library of labeled clones that is followed byligation, real time monitoring of the incorporation of labelednucleotides during a polymerization step, polony sequencing, and SOLiDsequencing. Sequencing of the separated molecules has been carried outby sequential or single extension reactions using polymerases or ligasesas well as by single or sequential differential hybridizations withlibraries of probes. These reactions have been performed on many clonalsequences in parallel including demonstrations in current commercialapplications of over 100 million sequences in parallel. These sequencingapproaches can thus be used to study the repertoire of T-cell receptor(TCR) and/or B-cell receptor (BCR).

In one aspect of the invention, high-throughput methods of sequencingare employed that comprise α step of spatially isolating individualmolecules on a solid surface where they are sequenced in parallel. Suchsolid surfaces may include nonporous surfaces (such as in Solexasequencing, e.g. Bentley et al, Nature, 456: 53-59 (2008) or CompleteGenomics sequencing, e.g. Drmanac et al, Science, 327: 78-81 (2010)),arrays of wells, which may include bead- or particle-bound templates(such as with 454, e.g. Margulies et al, Nature, 437: 376-380 (2005) orIon Torrent sequencing, U.S. patent publication 2010/0137143 or2010/0304982), micromachined membranes (such as with SMRT sequencing,e.g. Eid et al, Science, 323: 133-138 (2009)), or bead arrays (as withSOLiD sequencing or polony sequencing, e.g. Kim et al, Science, 316:1481-1414 (2007)).

In another aspect, such methods comprise amplifying the isolatedmolecules either before or after they are spatially isolated on a solidsurface. Prior amplification may comprise emulsion-based amplification,such as emulsion PCR, or rolling circle amplification. Of particularinterest is Solexa-based sequencing where individual template moleculesare spatially isolated on a solid surface, after which they areamplified in parallel by bridge PCR to form separate clonal populations,or clusters, and then sequenced, as described in Bentley et al (citedabove) and in manufacturer's instructions (e.g. TruSeq™ SamplePreparation Kit and Data Sheet, Illumina, Inc., San Diego, Calif.,2010); and further in the following references: U.S. Pat. Nos.6,090,592; 6,300,070; 7,115,400; and EP0972081B1; which are incorporatedby reference. In one embodiment, individual molecules disposed andamplified on a solid surface form clusters in a density of at least 10⁵clusters per cm²; or in a density of at least 5×10⁵ per cm²; or in adensity of at least 10⁶ clusters per cm². In one embodiment, sequencingchemistries are employed having relatively high error rates. In suchembodiments, the average quality scores produced by such chemistries aremonotonically declining functions of sequence read lengths.

In one aspect, a sequence-based clonotype profile of an individual isobtained using the following steps: (a) obtaining a nucleic acid sample,for example, a sample containing T-cells of the individual; (b)spatially isolating individual molecules derived from such nucleic acidsample, the individual molecules comprising at least one templategenerated from a nucleic acid in the sample, which template comprises asomatically rearranged region or a portion thereof, each individualmolecule being capable of producing at least one sequence read; (c)sequencing said spatially isolated individual molecules to providesequence reads; and (d) determining abundances of different sequences ofthe nucleic acid molecules from the nucleic acid sample to generate theclonotype profile. In some embodiments, the step of sequencing includescoalescing at least a plurality of sequence reads to form eachclonotype. As described more fully below, such a step of coalescing is aprocess of combining sequence reads with error rates (for example, fromsequencing and/or amplification errors) to produce clonotypes that arecorrect with a high degree of likelihood, such as with a 99% confidencelevel.

In one aspect, for each sample from an individual, the sequencingtechnique used in the methods of the invention generates sequences ofleast 1000 sequence reads per run; in another aspect, such techniquegenerates sequences of at least 10,000 sequence reads per run; inanother aspect, such technique generates sequences of at least 100,000sequence reads per run; in another aspect, such technique generatessequences of at least 500,000 sequence reads per run; and in anotheraspect, such technique generates sequences of at least 1,000,000sequence reads per run. From such sequence reads clonotypes aredetermined, for example, as described below, or as disclosed in Fahamand Willis (described above).

The sequencing techniques used in the methods generate sequence readshaving lengths of at least 30 nucleotides. In some embodiments, a stepof sequencing generates sequence reads having lengths of at least 50nucleotides; and in some embodiments, a step of sequencing generatessequence reads having lengths of at least 100 nucleotides.

E. Clonotype Determination from Sequence Data

Constructing clonotypes from sequence read data depends in part on thesequencing method used to generate such data, as the different methodshave different expected read lengths and data quality. In one approach,a Solexa sequencer is employed to generate sequence read data foranalysis. In one embodiment, a sample is obtained that provides at least0.5-1.0×10⁶ lymphocytes to produce at least 1 million templatemolecules, which after optional amplification may produce acorresponding one million or more clonal populations of templatemolecules (or clusters). For most high throughput sequencing approaches,including the Solexa approach, such over sampling at the cluster levelis desirable so that each template sequence is determined with a largedegree of redundancy to increase the accuracy of sequence determination.For Solexa-based implementations, preferably the sequence of eachindependent template is determined 10 times or more. For othersequencing approaches with different expected read lengths and dataquality, different levels of redundancy may be used for comparableaccuracy of sequence determination. Those of ordinary skill in the artrecognize that the above parameters, e.g. sample size, redundancy, andthe like, are design choices related to particular applications.

In one aspect of the invention, sequences of clonotypes (including butnot limited to those derived from TCR alpha, TCR beta, TCR gamma, and/orTCR delta, may be determined by combining information from a pluralityof sequence reads sequence reads, for example, along the V(D)J regionsof the selected chains. In another aspect, sequences of clonotypes aredetermined by combining information from a plurality of sequence reads.Such pluralities of sequence reads may include one or more sequencereads along a sense strand (i.e. “forward” sequence reads) and one ormore sequence reads along its complementary strand (i.e. “reverse”sequence reads).

Sequence reads of the invention may have a wide variety of lengths,depending in part on the sequencing technique being employed. Forexample, for some techniques, several trade-offs may arise in itsimplementation, for example, (i) the number and lengths of sequencereads per template and (ii) the cost and duration of a sequencingoperation. In one embodiment, sequence reads are in the range of from 20to 400 nucleotides; in another embodiment, sequence reads are in a rangeof from 30 to 200 nucleotides; in still another embodiment, sequencereads are in the range of from 30 to 120 nucleotides. In one embodiment,2 to 1000 sequence reads are generated for determining the sequence ofeach clonotype; in another embodiment, 2 to 100 sequence reads aregenerated for determining the sequence of each clonotype; and in anotherembodiment, 2 to 10 sequence reads are generated for determining thesequence of each clonotype; and in still another embodiment, at least 10sequence reads are generated for determining the sequence of eachclonotype. In the foregoing embodiments, the numbers given are exclusiveof sequence reads used to identify samples from different individuals.The lengths of the various sequence reads used in the embodimentsdescribed below may also vary based on the information that is sought tobe captured by the read; for example, the starting location and lengthof a sequence read may be designed to provide the length of an NDNregion as well as its nucleotide sequence; thus, sequence reads spanningthe entire NDN region are selected. In other aspects, one or moresequence reads that in combination (but not separately) encompass a Dand/or NDN region are sufficient.

In another aspect of the invention, sequences of clonotypes aredetermined in part by aligning sequence reads to one or more V regionreference sequences and one or more J region reference sequences, and inpart by base determination without alignment to reference sequences,such as in the highly variable NDN region. A variety of alignmentalgorithms may be applied to the sequence reads and reference sequences.For example, guidance for selecting alignment methods is available inBatzoglou, Briefings in Bioinformatics, 6: 6-22 (2005), which isincorporated by reference. In one aspect, whenever V reads or C reads(as mentioned above) are aligned to V and J region reference sequences,a tree search algorithm may be employed, e.g. as described generally inGusfield (cited above) and Cormen et al, Introduction to Algorithms,Third Edition (The MIT Press, 2009).

In another aspect, an end of at least one forward read and an end of atleast one reverse read overlap in an overlap region (e.g. 308 in FIG.14A), so that the bases of the reads are in a reverse complementaryrelationship with one another. Thus, for example, if a forward read inthe overlap region is “5′-acgttgc”, then a reverse read in a reversecomplementary relationship is “5′-gcaacgt” within the same overlapregion. In one aspect, bases within such an overlap region aredetermined, at least in part, from such a reverse complementaryrelationship. That is, a likelihood of a base call (or a related qualityscore) in a prospective overlap region is increased if it preserves, oris consistent with, a reverse complementary relationship between the twosequence reads.

In one aspect, clonotypes of TCR beta and IgH chains (illustrated inFIG. 14A) are determined by at least one sequence read starting in its Jregion and extending in the direction of its associated V region(referred to herein as a “C read” (304)) and at least one sequence readstarting in its V region and extending in the direction of itsassociated J region (referred to herein as a “V read” (306)). Overlapregion (308) may or may not encompass the NDN region (315) as shown inFIG. 14A. Overlap region (308) may be entirely in the J region, entirelyin the NDN region, entirely in the V region, or it may encompass a Jregion-NDN region boundary or a V region-NDN region boundary, or bothsuch boundaries (as illustrated in FIG. 3B). Typically, such sequencereads are generated by extending sequencing primers, e.g. (302) and(310) in FIG. 14A, with a polymerase in a sequencing-by-synthesisreaction, e.g. Metzger, Nature Reviews Genetics, 11: 31-46 (2010);Fuller et al, Nature Biotechnology, 27: 1013-1023 (2009). The bindingsites for primers (302) and (310) are predetermined, so that they canprovide a starting point or anchoring point for initial alignment andanalysis of the sequence reads. In one embodiment, a C read ispositioned so that it encompasses the D and/or NDN region of the TCRbeta or IgH chain and includes a portion of the adjacent V region, e.g.as illustrated in FIGS. 14A and 14B. In one aspect, the overlap of the Vread and the C read in the V region is used to align the reads with oneanother. In other embodiments, such alignment of sequence reads is notnecessary, e.g. with TCR beta chains, so that a V read may only be longenough to identify the particular V region of a clonotype. This latteraspect is illustrated in FIG. 14B. Sequence read (330) is used toidentify a V region, with or without overlapping another sequence read,and another sequence read (332) traverses the NDN region and is used todetermine the sequence thereof. Portion (334) of sequence read (332)that extends into the V region is used to associate the sequenceinformation of sequence read (332) with that of sequence read (330) todetermine a clonotype. For some sequencing methods, such as base-by-baseapproaches like the Solexa sequencing method, sequencing run time andreagent costs are reduced by minimizing the number of sequencing cyclesin an analysis. Optionally, as illustrated in FIG. 14A, amplicon (300)is produced with sample tag (312) to distinguish between clonotypesoriginating from different biological samples, e.g. different patients.Sample tag (312) may be identified by annealing a primer to primerbinding region (316) and extending it (314) to produce a sequence readacross tag (312), from which sample tag (312) is decoded.

Reducing a set of reads for a given sample to a set of distinctclonotypes and recording the number of reads for each clonotype would bea trivial computational problem if sequencing technology was error free.However, in the presence of sequencing errors, each genuine clonotype issurrounded by a “cloud” of reads with varying numbers of errors withrespect to the its sequence. The “cloud” of sequencing errors drops offin density as the distance increases from the clonotype in sequencespace. A variety of algorithms are available for converting sequencereads into clonotypes. In one approach, coalescing of sequence reads(that is, merging candidate clonotypes determined to have one or moresequencing errors) depends on at least three factors: the number ofsequences obtained for each of the clonotypes being compared; the numberof bases at which they differ; and the sequencing quality score at thepositions at which they are discordant. A likelihood ratio may beconstructed and assessed that is based on the expected error rates andbinomial distribution of errors. For example, two clonotypes, one with150 reads and the other with 2 reads with one difference between them inan area of poor sequencing quality will likely be coalesced as they arelikely to be generated by sequencing error. On the other hand twoclonotypes, one with 100 reads and the other with 50 reads with twodifferences between them are not coalesced as they are considered to beunlikely to be generated by sequencing error.

In one embodiment of the invention, the algorithm described below may beused for determining clonotypes from sequence reads. In one approach,sequence reads are first converted into candidate clonotypes. Such aconversion depends on the sequencing platform employed. For platformsthat generate high Q score long sequence reads, the sequence read or aportion thereof may be taken directly as a candidate clonotype. Forplatforms that generate lower Q score shorter sequence reads, somealignment and assembly steps may be required for converting a set ofrelated sequence reads into a candidate clonotype. For example, forSolexa-based platforms, in some embodiments, candidate clonotypes aregenerated from collections of paired reads from multiple clusters, e.g.10 or more, as mentioned above.

The cloud of sequence reads surrounding each candidate clonotype can bemodeled using the binomial distribution and a simple model for theprobability of a single base error. This latter error model can beinferred from mapping V and J segments or from the clonotype findingalgorithm itself, via self-consistency and convergence. A model isconstructed for the probability of a given “cloud” sequence Y with readcount C₂ and E errors (with respect to sequence X) being part of a trueclonotype sequence X with perfect read count C₁ under the null modelthat X is the only true clonotype in this region of sequence space. Adecision is made whether or not to coalesce sequence Y into theclonotype X according the parameters C₁, C₂, and E. For any given C₁ andE a max value C₂ is pre-calculated for deciding to coalesce the sequenceY. The max values for C₂ are chosen so that the probability of failingto coalesce Y under the null hypothesis that Y is part of clonotype X isless than some value P after integrating over all possible sequences Ywith error E in the neighborhood of sequence X. The value P is controlsthe behavior of the algorithm and makes the coalescing more or lesspermissive.

If a sequence Y is not coalesced into clonotype X because its read countis above the threshold C₂ for coalescing into clonotype X then itbecomes a candidate for seeding separate clonotypes (such as withcandidate clonotype 2. An algorithm implementing such principles wouldalso make sure that any other sequences Y2, Y3, etc. which are ‘nearer’to this sequence Y (that had been deemed independent of X) are notaggregated into X. This concept of “nearness” includes both error countswith respect to Y and X and the absolute read count of X and Y, i.e. itis modeled in the same fashion as the above model for the cloud of errorsequences around clonotype X. In this way ‘cloud’ sequences can beproperly attributed to their correct clonotype if they happen to be‘near’ more than one clonotype.

In some embodiments, an algorithm proceeds in a top down fashion bystarting with the sequence X with the highest read count. This sequenceseeds the first clonotype. Neighboring sequences are either coalescedinto this clonotype if their counts are below the pre-calculatedthresholds (see above), or left alone if they are above the threshold or“closer” to another sequence that was not coalesced. After searching allneighboring sequences within a maximum error count, the process ofcoalescing reads into clonotype X is finished. Its reads and all readsthat have been coalesced into it are accounted for and removed from thelist of reads available for making other clonotypes. The next sequenceis then moved on to with the highest read count. Neighboring reads arecoalesced into this clonotype as above and this process is continueduntil there are no more sequences with read counts above a giventhreshold, e.g. until all sequences with more than 1 count have beenused as seeds for clonotypes.

As mentioned above, in another embodiment of the above algorithm, afurther test may be added for determining whether to coalesce acandidate sequence Y into an existing clonotype X, which takes intoaccount quality score of the relevant sequence reads. The averagequality score(s) are determined for sequence(s) Y (averaged across allreads with sequence Y) were sequences Y and X differ. If the averagescore is above a predetermined value then it is more likely that thedifference indicates a truly different clonotype that should not becoalesced and if the average score is below such predetermined valuethen it is more likely that sequence Y is caused by sequencing errorsand therefore should be coalesced into X. Successful implementation ofthe above algorithm for coalescing candidate clonotypes is dependentupon having an efficient way of finding all sequences with less than Eerrors (i.e. less than some sequence distance measure) from some inputsequence X. One approach is using a sequence tree. The implementation ofsuch trees has some unusual features in that the nodes of the tree arenot restricted to being single letters of the DNA sequences of thecandidate clonotypes. The nodes can have arbitrarily long sequences,which allows for a more efficient use of computer memory.

For example, all of the reads of a given sample are placed into thesequence tree. Each leaf nodes holds pointers to its associated reads. Aunique sequence of a candidate clonotype is retrieved by traversingbackwards in the tree from the leaf to the root node. The first sequenceis placed into a simple tree with one root node and one leaf node thatcontains the full sequence of the read. Sequences are next added one byone. For each added sequence either a new branch is formed at the lastpoint of common sequence between the read and the existing tree or addthe read to an existing leaf node if the tree already contains thesequence. Having placed all the reads into the tree it is easy to usethe tree for the following purposes: 1) Finding the highest read count:sorting leaf nodes by read count allows one to find the leaf node (i.e.sequence) with the most reads, and successively lower numbers of reads;2) Finding neighboring leafs: for any sequence all paths through thetree which have less than X errors with respect to this sequence aresearchable. A path is started at the root and branch this path intoseparate paths proceeding along the tree. The current error count ofeach path as proceeding along the tree is noted. When the error countexceeds the max allowed errors the given path is terminated. In this waylarge parts of the tree are pruned as early as possible. This is anefficient way of finding all paths (i.e. all leafs) within X errors fromany given sequence.

F. TCR Beta Repertoire Analysis

In this approach, TCR beta chains are analyzed and clonotypes aredetermined. The analysis includes amplification, sequencing, andanalyzing the TCR beta sequences. One primer is complementary to acommon sequence in C beta1 and C beta2, and there are 34 V primerscapable of amplifying all 48 V segments. C beta1 or C beta2 differ fromeach other at position 10 and 14 from the J/C junction. The primer for Cbeta1 and C beta2 ends at position 16 bp and has no preference for Cbeta1 or C beta2. The 34 V primers are modified from an original set ofprimers disclosed in Van Dongen et al, U.S. Patent ApplicationPublication No. 2006/0234234, which is incorporated herein by reference.The modified primers are disclosed in Faham et al, U.S. PatentApplication Publication No. 2010/0151471, which is also incorporatedherein by reference.

The Illumina Genome Analyzer is used to sequence the amplicon producedby the above primers. A two-stage amplification is performed onmessenger RNA transcripts (200), as illustrated in FIGS. 13A-13B, thefirst stage employing the above primers and a second stage to add commonprimers for bridge amplification and sequencing. As shown in FIG. 13A, aprimary PCR is performed using on one side a 20 bp primer (202) whose 3′end is 16 bases from the J/C junction (204) and which is perfectlycomplementary to C beta1 (203) and the two alleles of C beta2. In the Vregion (206) of RNA transcripts (200), primer set (212) is providedwhich contains primer sequences complementary to the different V regionsequences (34 in one embodiment). Primers of set (212) also contain anon-complementary tail (214) that produces amplicon (216) having primerbinding site (218) specific for P7 primers (220). After a conventionalmultiplex PCR, amplicon (216) is formed that contains the highly diverseportion of the J(D)V region (206, 208, and 210) of the mRNA transcriptsand common primer binding sites (203 and 218) for a secondaryamplification to add a sample tag (221) and primers (220 and 222) forcluster formation by bridge PCR. In the secondary PCR, on the same sideof the template, a primer (222 in FIG. 13B and referred to herein as“C10-17-P5”) is used that has at its 3′ end the sequence of the 10 basesclosest to the J/C junction, followed by 17 bp with the sequence ofpositions 15-31 from the JC junction, followed by the P5 sequence (224),which plays a role in cluster formation by bridge PCR in Solexasequencing. (When the C10-17-P5 primer (222) anneals to the templategenerated from the first PCR, a 4 bp loop (position 11-14) is created inthe template, as the primer hybridizes to the sequence of the 10 basesclosest to the J/C junction and bases at positions 15-31 front the J/Cjunction. The looping of positions 11-14 eliminates differentialamplification of templates carrying C beta1 or C beta2. Sequencing isthen done with a primer complementary to the sequence of the 10 basesclosest to the J/C junction and bases at positions 15-31 from the J/Cjunction (this primer is called C′). C10-17-P5 primer can be HPLCpurified in order to ensure that all the amplified material has intactends that can be efficiently utilized in the cluster formation.)

In FIG. 13A, the length of the overhang on the V primers (212) ispreferably 14 bp. The primary PCR is helped with a shorter overhang(214). Alternatively, for the sake of the secondary PCR, the overhang inthe V primer is used in the primary PCR as long as possible because thesecondary PCR is priming from this sequence. A minimum size of overhang(214) that supports an efficient secondary PCR was investigated. Twoseries of V primers (for two different V segments) with overhang sizesfrom 10 to 30 with 2 bp steps were made. Using the appropriate syntheticsequences, the first PCR was performed with each of the primers in theseries and gel electrophoresis was performed to show that all amplified.

As illustrated in FIG. 13A, the primary PCR uses 34 different V primers(212) that anneal to V region (206) of RNA templates (200) and contain acommon 14 bp overhang on the 5′ tail. The 14 bp is the partial sequenceof one of the Illumina sequencing primers (termed the Read 2 primer).The secondary amplification primer (220) on the same side includes P7sequence, a tag (221), and Read 2 primer sequence (223) (this primer iscalled Read2_tagX_P7). The P7 sequence is used for cluster formation.Read 2 primer and its complement are used for sequencing the V segmentand the tag respectively. A set of 96 of these primers with tagsnumbered 1 through 96 are created (see below). These primers are HPLCpurified in order to ensure that all the amplified material has intactends that can be efficiently utilized in the cluster formation.

As mentioned above, the second stage primer, C-10-17-P5 (222, FIG. 2B)has interrupted homology to the template generated in the first stagePCR. The efficiency of amplification using this primer has beenvalidated. An alternative primer to C-10-17-P5, termed CsegP5, hasperfect homology to the first stage C primer and a 5′ tail carrying P5.The efficiency of using C-10-17-P5 and CsegP5 in amplifying first stagePCR templates was compared by performing real time PCR. In severalreplicates, it was found that PCR using the C-10-17-P5 primer had littleor no difference in efficiency compared with PCR using the CsegP5primer.

Amplicon (230) resulting from the 2-stage amplification illustrated inFIGS. 13A-13C has the structure typically used with the Illuminasequencer as shown in FIG. 13C. Two primers that anneal to the outmostpart of the molecule, Illumina primers P5 and P7 are used for solidphase amplification of the molecule (cluster formation). Three sequencereads are done per molecule. The first read of 100 bp is done with theC′ primer, which has a melting temperature that is appropriate for theIllumina sequencing process. The second read is 6 bp long only and issolely for the purpose of identifying the sample tag. It is generatedusing a tag primer provided by the manufacturer (Illumina). The finalread is the Read 2 primer, also provided by the manufacturer (Illumina).Using this primer, a 100 bp read in the V segment is generated startingwith the 1st PCR V primer sequence.

II. Identifying Paired T-Cell Receptor Chains without Antigen-SpecificSelection

In one aspect, the invention provides methods for matching pairs ofimmune receptor chains from populations of their encoding nucleic acidsthat have been sequenced. In accordance with one embodiment of theinvention, nucleic acid populations encoding repertoires of heavy chainvariable regions and light chain variable regions are sequenced so thattwo separate lists of sequences are formed without any correspondencebetween members of each list. This may be achieved by carrying outseparate sequencing operations, or runs, for each chain, or it may beaccomplished by carrying out a single sequence run with the nucleicacids tagged according to the identity of the type of chain it encodes.In accordance with another embodiment of the invention, nucleic acidpopulations encoding repertoires of T cell receptor alpha (TCR alpha)chains and T cell receptor beta (TCR beta) chains are sequenced, so thattwo separate lists of sequences are formed without any correspondencebetween members of each list. In accordance with another embodiment ofthe invention, nucleic acid populations encoding repertoires of T cellreceptor gamma (TCR gamma) chains and T cell receptor delta (TCR delta)chains are sequenced, so that two separate lists of sequences are formedwithout any correspondence between members of each list. As above, thismay be achieved by carrying out separate sequencing runs for each chain,or it may be accomplished by carrying out a single sequence run with thenucleic acids tagged according to the identity of the type of chain itencodes (that is, either TCR alpha and TCR beta, or TCR gamma and TCRdelta, respectively). In the latter embodiments, two approaches may befollowed for matching or pairing TCR alpha and TCR beta (or TCR gammaand TCR delta) chains into chains that are functional, for example,because they originate from the same T cell. In a first approach, thefrequencies of each encoding nucleic acid are determined and TCR alphachains and TCR beta chains whose encoding nucleotide sequences have thesame frequencies are paired to form a functional, or reconstituted, TCR.TCR gamma and TCR delta chains may be matched by the same process. In asecond approach, which is applicable to matching all three types ofimmune receptor pairs, a lymphocyte population is repeatedly dividedinto a plurality of subsets. Such subsets may be obtained by aliquotinga tissue sample into separate reaction vessels or chambers. Separatelyfrom each of a portion, or subpopulation, of the subsets, nucleic acidsencoding the two different immune receptor chains are extracted andsequenced, so that two separate lists of sequences are formed withoutany correspondence between members of each list. As described above,this may be achieved by carrying out separate sequencing runs for eachchain, or it may be accomplished by carrying out a single sequence runwith the nucleic acids tagged according to the identity of the type ofchain it encodes. To illustrate by an example, if a sample containing Tcells or B cells is aliquoted into 100 sub-samples, so that on averageeach aliquot contains a subset consisting of about 1/100 of the totalnumber of T cells or B cells in the original sample, then 20 suchsubsets may be randomly selected as a portion of the total number ofsubsets. (Such portion could be any number greater than one and lessthan 100, although as described more fully below, a number in the rangeof from 10 to 20 is a good trade-off between amount of sequencingrequired and likelihood of identifying receptor pairs present at afrequency of interest). In one embodiment, a plurality of subsets is inthe range of from 20 to 2000 and a portion of subsets thereof is in therange of from 10 to 50. In another embodiment, a portion of subsets isin the range of from 10 to 20. Examples of the above embodiments areillustrated in FIGS. 12A and 12B.

As illustrated in FIG. 12A, nucleic acid (which may be DNA or RNA) isextracted from a sample containing T cells (100), after which inseparate reaction volumes, primers (102) specific for a nucleic acidsencoding TCR alpha chains (or a portion thereof) and primers (104)specific for nucleic acids encoding TCR beta chains (or a portionthereof) are combined under conditions that allow the respective nucleicacid populations to be amplified, e.g. by a two-stage polymerase chainreaction (PCR), such as disclosed by Faham and Willis (cited above).Guidance and disclosures for selecting such primers and carrying outsuch reactions are described extensively in the molecular immunologyliterature and below (for TCR beta and IgH) and in references such as,Yao et al, Cellular and Molecular Immunology, 4: 215-220 (2007)(for TCRalpha), the latter reference being incorporated herein by reference. Inone embodiment, amplicons (106) and (108) produced by a two-stage PCRare ready for sequence analysis using a commercially available nextgeneration sequencer, such as MiSeq Personal Sequencer (Illumina, SanDiego, Calif.). After nucleotide sequences have been determined (107)and (109), databases or tables (110 and 112, respectively) are obtained.Like sequences may be counted and frequency versus sequence plots (114and 116) are constructed. Reconstituted TCRs may be determined bymatching (118) TCR alpha chains and TCR beta chains with identicalfrequencies or with frequencies having the same rank ordering. Clearly,this embodiment of the method works most efficiently when frequencies ofdifferent TCR alpha chains and TCR beta chains are not too closetogether, i.e. are distinct, even taking into account experimentalerror.

Once a pair of clonotype sequences having equal (or equally ranked)frequencies are identified full length sequences encoding each chain maybe reconstructed from the known constant and variable regions usingconventional techniques for genetic manipulation and expression, e.g.Walchli et al, PLosOne, 6(11): e27930 (2011); or the like.

Greater accuracy in the determination of receptor chain frequencies maybe obtained in a variation of the above embodiment, which may be seen inreference to FIGS. 13A and 13B where RNA encoding TCR beta is amplifiedin a two-staged PCR. As described more fully below, primer (202) andprimer set (212) are used in a first stage amplification to attachcommon primer binding site (214) to all the nucleic acids encoding TCRbetas. FIG. 13B illustrates the components of a second stageamplification for generating more material and for attaching primerbinding sites P5 (222) and P7 (220) which are used in cluster formation(via bridge PCR) in the Solexa-based sequencing protocol. Primer P7(220) may also include sample tag (221) for multiplexing up to 96samples for concurrent sequencing in the same run, e.g. Illuminaapplication note 770-2008-011 (2008). A different type of tag in thesame primer may be used to increase the accuracy of the determination ofreceptor chain frequencies. In this embodiment, primer P7 is modified toinclude a highly diverse tag set, so that instead of 96 tags, primer P7is engineered to have 10,000 distinct tags, or more. In other words,primer P7 is a mixture of 10,000 or more distinct oligonucleotides eachhaving an identical template binding region, a distinct tag sequence,and an identical 5′ tail portion (e.g., (223) in FIG. 13B). With thisarrangement, any subset of nucleic acids encoding the same receptorchain (e.g. less than 100) will receive a different tag with highprobability. Such a process of pairing members of a small set of nucleicacids with a much larger set of tags for counting, labeling, sortingpurposes is well known and is disclosed in various forms in thefollowing references that are incorporated by reference, U.S. Pat. Nos.6,172,214; 7,537,897; International PCT Publication No. WOUS2005/111242; Brenner et al, Proc. Natl. Acad. Sci., 97: 1665-1670(2000); Casbon et al, Nucleic Acids Research, 39(12): e81 (2011); Fu etal, Proc. Natl. Acad. Sci., 108: 9026-9031 (2011).

Construction of sets of minimally cross-hybridizing oligonucleotide tag,or tags with other useful properties, is disclosed in the followingexemplary references, which are incorporated by reference: Brenner, U.S.Pat. No. 6,172,214; Morris et al, U.S. patent publication 2004/0146901;Mao et al, U.S. patent publication 2005/0260570; and the like.Preferably, the tag set should be at least 100 times (or more) the sizeof the set of nucleic acids to be labeled if all nucleic acids are toreceive a unique tag with high probability. For immune receptor chains,in one embodiment, the number of distinct tags is in the range of from10,000 to 100,000; in another embodiment, the number of distinct tags isin the range of from 10,000 to 50,000; and in another embodiment, thenumber of distinct tags is in the range of from 10,000 to 20,000. Asdisclosed in U.S. Pat. No. 6,172,214, such large mixtures ofoligonucleotide tags may be synthesized by combinatorial methods;alternatively, primers containing unique tags may be synthesizedindividually by non-combinatorial methods, such as disclosed by Clearyet al, Nature Methods, 1: 241-248 (2004); York et al, Nucleic AcidsResearch, 40(1): e4 (2012); LeProust et al, Nucleic Acids Research,38(8): 2522-2540 (2010); and the like.

In one aspect, the above embodiment may be carried out by the followingsteps: (a) obtaining a sample containing T cells; (b) determiningnucleotide sequences of TCR alpha chains of T cells from the sample,each TCR alpha chain having a frequency of occurrence in the sample; (c)determining nucleotide sequences of TCR beta chains of T cells from thesample, each TCR beta chain having a frequency of occurrence in thesample; and (d) identifying paired TCR alpha chains and TCR beta chainsas those having the same frequency within the sample. Frequencies of therespective TCR alpha chains and TCR beta chains may be determined fromthe tabulations of encoding nucleic acids, or clonotypes. Alternatively,frequencies of the respective TCR alpha chains and TCR beta chains maybe determined from the tabulations of polypeptides encoded by theclonotypes. As mentioned above, clonotype frequencies may be determinedby counting clonotypes directly or indirectly by using a tagging schemeas described above.

FIG. 12B illustrates another embodiment for identifying matchingreceptor subunits which may be applied to either TCRs or BCRs and whichmay be used even when receptor frequencies among subunit chains areclose or indistinguishable, whether because of experimental error orotherwise. Starting with a sample containing lymphocytes (149), whichmay be either T cells or B cells, subsets are formed by separating orpartitioning the sample into a plurality of subsets (152), 1 through K(in the figure). In some embodiments, only a portion of the K subset areanalyzed; thus, it is not necessary to actually form all K subsets. Onemay form subsets of only the portion that are actually analyzed. Forexample, if the sample has a volume of 100 μL and K=100, but only aportion consisting of 20 subset is to be analyzed, then only twenty 1 μLsubsets need be formed. From each subset (152) nucleic acids encodingeach different immune receptor chain (TCR alpha and TCR beta being shownunder subset 1) are sequenced, thereby forming pairs of lists, forexample, (162), (164), (166) and (168) for subsets 1, 2 . . . K−1, K,respectively. Each pair of such lists contains a first list ofnucleotide sequences of a first immune receptor chain, e.g. list (154)for TCR alpha of subset 1, and a second list of nucleotide sequences ofa second immune receptor chain, e.g. list (156) for TCR beta ofsubset 1. In one embodiment, the number of subsets, K, is a number inthe range of from 5 to 500; in another embodiment, K is a number in therange of from 10 to 100; in another embodiment, K is a number in therange of from 20 to 50. In some embodiments, a portion of subsetsanalyzed is 10 or fewer subsets; in other embodiments, a portion ofsubsets analyzed is 20 or fewer subsets; in other embodiments, a portionof subsets analyzed is at least five percent of the subsets; in otherembodiments, a portion of subsets analyzed is at least ten percent ofthe subsets; in other embodiments, a portion of subsets analyzed is atleast twenty percent of the subsets.

Each kind of lymphocyte in sample, e.g. lymphocyte (150), is present inthe sample at a particular frequency. The distribution of lymphocytesinto the subsets is readily approximated by a binomial model; thus, foran arbitrary lymphocyte (for example (150)) having a particularclonotype, (a) its frequency in the sample, (b) the total number oflymphocytes in the sample, and (c) the number of subsets may be relatedto the expectation of finding at least one of the particular lymphocytein a predetermined fraction of subsets. This relationship may beexpressed as follows: r=(1−f)^((N/K)), where r is the fraction ofsubsets containing at least one of the particular lymphocyte, f is thefrequency of the particular lymphocyte in the sample, N is the totalnumber of lymphocytes in the sample, and K is the number of subsets.Thus, if one sets r=1/2 and takes N as a constant, then one may selectsuccessive values of K so that lymphocytes of different frequencies arepresent in about half of the subsets. Other values of r could beselected, but r=1/2 provides results with the highest statistical power,thus the value r≈ 1/2 is preferred. Once such lists are obtained theyare examined to identify pairs of first and second nucleotide sequencesthat either occur in a subset together or are both absent from a subset.By way of example, the members of pair (158) appear in lists (164) ofsubset 2 and in lists (166) of subset K-1, but neither member of thepair appears in lists (162) or (168) of subsets 1 and K, either alone ortogether. This of course reflects the presence or absence of theparticular lymphocyte that is in subsets 2 and K−1, but is absent fromsubsets 1 and K, such as lymphocyte (150). Such a pattern confirms thatthe members of pair (158) go together and correspond to the chains of afunctional immune receptor. Other lymphocytes in sample (149) may bepresent in approximately the same frequency, such as lymphocyte (153).However, the probability that at least one of lymphocyte (153) willoccur in exactly the same subsets as lymphocyte (150) is extremely low,especially if r is approximately one half and the portion of the Ksubsets analyzed is in the range of from 10 to 20, or more.

In one aspect of the invention, matched first and second chains oflymphocytes from a succession of frequency classes may be determined bycarrying out the above process repeatedly for different values of K. Forexample, a 1 mL sample of peripheral blood of a normal individualcontains about 1-4.8×10⁶ lymphocytes of which about 10-15 percent are Bcells, about 70-85 percent are T cells and about 10 percent are NKcells; thus, the 1 mL sample may contain from about 7×10⁵ to about 4×10⁶T cells. If the number of T lymphocytes in a 1 mL sample is N=10⁶, thenmatching TCR chains of T cells of the following frequencies are matchedby identifying those that appear together in fifty percent of thesubsets and not at all in the other fifty percent of subsets:

Frequency Number of Subsets Volume (μL) .001 1443 0.7 .0005 722 1.4.0001 144 6.9 .00005 72 13.9

As mentioned above, not all the subsets at a particular frequency needbe analyzed. If there are a large number of lymphocytes that havefrequencies at or close to a selected frequency, e.g. f=0.001, they mayall be resolved by taking a larger and larger portion of the totalnumber of subsets until every pair that appears together in fiftypercent of the subsets can be distinguished from every other pair at thesame frequency. This is because the probability of two differentlymphocytes occurring in exactly the same subsets of the fifty percentbecomes infinitesimal as the portion of subsets is increased.

III. Identifying Paired and Unpaired T-Cell Receptor Chains withAntigen-Specific Selection

In some embodiments, the invention is directed to identifyingantigen-specific T cells by one or a pair of immune receptor chains,such as TCR alpha, or TCR beta, or TCR alpha and TCR beta together; orTCR delta, or TCR gamma, or TCR delta and TCR gamma together. In someembodiments, the nucleotide sequence encoding a single immune receptorchain, such as TCR beta, is used to identify antigen-specific T cells.Sometimes such nucleotide sequences are referred to herein as a“clonotype,” although clonotypes also may be ordered pairs of nucleotidesequences specific to a particular T cell, such as the nucleotidesequences encoding the T cell's TCR alpha and TCR beta chains, which maybe represented (for example) as (S_(α), S_(β)), or like notation, whereS_(α) is a sequence of a segment of TCR alpha and S_(β) is a sequence ofa segment of TCR beta, and as a pair they are a clonotype of the cellthey originate from.

Features of some embodiments of the invention are illustrated in FIG.12C. To a tissue sample (170) comprising T cells (173) is added antigen(171) under interaction conditions so that T cells specific for antigen(171) may interact with antigen (171). Such interaction may be direct orindirect. Direct interactions include binding of antigen (171) toantigen-specific T cells, binding of antigen peptide-multimer conjugatesto antigen-specific T cells, and the like. Peptide-multimer conjugates,such as tetramers, are well-known reagents to those of ordinary skill,e.g. Bousso, Microbes Infect. 2(4): 425-429 (2000); Klenerman et al,Nature Reviews Immunol., 2(4): 263-272 (2002); and the like. Indirectinteractions include presentation of antigen or antigen peptides toantigen-specific T cells by antigen presenting cells, such as, dendriticcells, artificial APCs, and the like. In some interactions,antigen-specific T cells may become activated T cells that mayproliferate and/or develop or express activation markers both of whichprovide means for selecting and/or enriching antigen-specific T cellsusing conventional techniques. Antigen (171) may comprise α wide varietyof compounds or compositions as discussed more fully below. Proteins andpeptides derived from one or more proteins are of special interest,particularly when the proteins are associated with cancers or infectiousdiseases, such as bacterial or virus infections. Antigen (171) may becombined with, exposed to, or added to, tissue sample (170) in a varietyof ways known in the art, e.g. Berzofsky et al, J. Clin. Investigation,113: 1515-1525 (2004). After combining antigen (171) with tissue sample(170) in a reaction mixture, antigen-specific T cells (173) andnon-antigen-specific T cells alike are exposed to antigen (171) withwhich they interact either directly or indirectly.

In some embodiments, antigen-specific T cells (173) are activated,possibly after a period of incubation with antigen (171). A period ofincubation may vary widely. In some embodiments, incubation may be foran interval of from a few minutes (for example, 10 minutes) to an houror more; in other embodiments, incubation may be for an interval of afew hours (for example, 2 hours) to 8 or more hours. In otherembodiments, antigen-specific T cells (173) interact with antigen bybinding to or forming complexes with antigen or antigen reagents, suchas antigen peptide-multimer conjugates, such that activation may nottake place. A step of exposing may include the step of incubating atissue sample with an antigen. For example, in the case of a proteinantigen and a tissue sample that comprises PBMCs, a step of exposing mayinclude combining the tissue sample with peptides derived from theprotein antigen such that dendritic cells in the tissue sample presentthe peptides to antigen-specific T cells in the tissue sample which, inturn, interact with the antigen-presenting dendritic cells and areactivated. After exposing T cells (173) to antigen so thatantigen-specific T cells interact with antigen, antigen-specific T cellsmay be selected (172) and/or enriched based on some feature resultingfrom the interaction, such as antigen peptide-multimer binding,activation markers induced, proliferation of the T cells, or the like.As mentioned above, the step of selecting (172) antigen-specific T cellsmay be alternatively a step of enriching antigen-specific T cells fromthe reaction mixture, and/or a step of separating antigen-specific Tcells from the reaction mixture, and/or a step of isolatingantigen-specific T cells from the reaction mixture. Afterantigen-specific T cells are enriched, separated, and/or isolated (172)their clonotypes are determined by sequencing a predetermined segment ofa recombined nucleic acid that encodes a portion of an immune receptor,such as TCR beta and/or TCR alpha.

A predetermined segment chosen may vary widely; in some embodiments, itencompasses all or a portion of a V(D)J region, so that clonotypes basedthereon have maximal diversity for unique identification of cell clones.Determination of clonotypes is described more fully below, but briefly,recombined nucleic acids encoding one or more selected immune receptors(such as TCR beta as shown in FIG. 12C) are sequenced (for example, byspatially isolating molecules thereof, amplifying such molecules, andcarrying out sequencing steps by a high-throughput sequencing chemistry,such as available with commercial next-generation DNA sequencers). As aresult of these sequencing steps, sequence reads (176) are producedwhich are used to determine clonotypes and clonotype frequencies ofantigen-specific T cells. Clonotypes and clonotype frequencies are alsodetermined either for T cells of the tissue sample (174) from sequencereads (178) or for non-antigen-specific T cells (175) from sequencereads (177). Non-antigen-specific T cells may be obtained from a two-waysorting procedure (for example, using FACS or MACS) based on T cellslabeled according to an interaction, such as, an interaction ofantigen-specific T cells with fluorescently labeled antigen peptidemultimers. These data may then be analyzed to identify clonotypesassociated with antigen-specific T cells, for example, as described inthe Example below and FIGS. 17-20 . Briefly, in some embodiments,antigen-specific T cells may be associated with clonotype frequenciesthat increase in the selected population of T cells relative tofrequencies of the same clonotype in populations of non-antigen specificT cells or in the population of T cells in tissue sample (170).

Exemplary steps for implementing this embodiment of the invention (i.e.,for determining clonotypes associated with antigen-specific T cells in atissue sample) may include the following: (a) exposing the T cells ofthe sample to an antigen so that T cells specific for the antigeninteract with the antigen; (b) sequencing recombined nucleic acidsencoding a T-cell receptor chain or a portion thereof from a sample of Tcells from the tissue sample to provide sequence reads from whichclonotypes are determined; (c) isolating antigen-specific T cells fromthe tissue sample based on their interaction with the antigen; (d)sequencing recombined nucleic acids encoding a T-cell receptor chain ora portion thereof from a sample of the isolated antigen-specific T cellsto provide sequence reads from which clonotypes are determined; and (e)determining antigen-specific T cells in the tissue sample as T cellswhose clonotype frequencies increase in the sample of isolated T cellsrelative to the frequencies of the same clonotypes in a sample of Tcells in the tissue sample.

In some embodiments, a step of exposing may be carried out by reactingunder interaction conditions an antigen with a tissue sample; in stillother embodiments, a step of exposing may be carried out by reactionunder activation conditions an antigen with a tissue sample. Asmentioned above the step of exposing for this and other embodiments mayvary widely, and its implementation may depend on the nature of thetissue sample and the nature of the antigen, as well as other factors.For example, if a tissue sample includes antigen-presenting cells, suchas dendritic cells, then exposing may include either addition of anantigen, such as a protein, directly to the tissue sample, or it mayinclude producing antigenic material from an antigen of interestfollowed by addition of the antigenic material. More efficient T cellactivation to a protein antigen, for example, may be accomplished byexposing a tissue sample to a set of overlapping peptides derived fromthe protein antigen of interest, using conventional techniques.Alternatively, artificial antigen-presenting compositions may be used inthe exposing step or its equivalent, e.g. Oelke et al, Nature Medicine,9(5): 619-624 (2003).

The step of exposing T cells in a tissue sample may include exposingsuch T cells to whole cells containing antigen, to gene-modified cellsexpressing antigen, to whole protein, to peptides derived from a proteinantigen, to viral vectors expressing an antigen, to antigen-modified, orloaded, dendritic cells. In some embodiments, a tissue sample is a bloodsample; in other embodiments, a tissue sample is a sample of peripheralblood mononuclear cells (PBMCs) derived from peripheral blood usingconventional techniques. In some embodiments the step of exposing may becarried out by reacting under activation conditions a tissue samplecomprising T cells with an antigen, where various activation conditionsare described above. In view of the wide variety of tissue samples andantigens, the step of exposing may be alternatively carried out by astep of reacting under activation conditions a tissue sample comprisingT cells with an antigen.

Further exemplary steps for implementing the above method may comprise:(a) reacting under activation conditions a tissue sample comprising Tcells to an antigen; (b) sorting from the tissue sample activated Tcells and un-activated T cells; (b) sequencing recombined nucleic acidsencoding a T-cell receptor chain or a portion thereof from a sample of Tcells from the activated T cells to provide sequence reads from whichclonotypes are determined; (c) sequencing recombined nucleic acidsencoding a T-cell receptor chain or a portion thereof from a sample of Tcells from the un-activated T cells to provide sequence reads from whichclonotypes are determined; and (d) determining antigen-specific T cellsin the tissue sample as T cells whose clonotype frequencies increase inthe sample of activated T cells relative to the frequencies of the sameclonotypes in the tissue sample or in a sample of un-activated T cells.Likewise, exemplary steps for implementing the above method maycomprise: (a) reacting under interaction conditions a tissue samplecomprising T cells with an antigen; (b) sorting T cells of the tissuesample into a first subset of T cells that form complexes with theantigen or antigen reagents thereof and into a second subset of T cellsthat do not form complexes with the antigen or antigen reagents thereof,(b) sequencing recombined nucleic acids encoding a T-cell receptor chainor a portion thereof from a sample of the first subset to providesequence reads from which clonotypes are determined; (c) sequencingrecombined nucleic acids encoding a T-cell receptor chain or a portionthereof from a sample of T cells from the tissue sample or the secondsubset to provide sequence reads from which clonotypes are determined;and (d) determining antigen-specific T cells in the tissue sample as Tcells whose clonotype frequencies increase in the sample of T cells ofthe first subset relative to the frequencies of the same clonotypes inthe tissue sample or in a sample of T cells from the second subset. Asused herein, the term “antigen reagents” means reagents derived from anantigen designed to bind to, or form complexes with, T cells whose TCRsare specific for the antigen. Exemplary antigen reagents include, butare not limited to, multimers conjugated with peptides derived from anantigen.

In some embodiments, the above method of determining antigen-specific Tcells in a tissue sample may be carried out by steps comprising: (a)reacting under activation conditions in a reaction mixture a tissuesample comprising T cells to an antigen or antigen reagents thereof; (b)sequencing recombined nucleic acids encoding a T-cell receptor chain ora portion thereof from a sample of T cells from the reaction mixtureprior to addition of the antigen to the reaction mixture to providesequence reads from which clonotypes are determined; (c) incubating thereaction mixture after addition of the antigen or antigen reagentthereof for a predetermined interval; (d) sequencing recombined nucleicacids encoding a T-cell receptor chain or a portion thereof from asample of T cells from the incubated reaction mixture to providesequence reads from which clonotypes are determined; (d) determiningantigen-specific T cells in the tissue sample as T cells whose clonotypefrequencies increase in the incubated reaction mixture relative to thefrequencies of the same clonotypes in the reaction mixture prior to theaddition of antigen. In some embodiments, a predetermined interval forincubation is usually greater than eight hours; in other embodiments, apredetermined interval may be greater than 24 hours; in furtherembodiments, a predetermined interval may be within a range of from 8hours to 72 hours.

In some embodiments, step of isolating antigen-specific T cells may besubstituted with either a step of separating a sample ofantigen-specific T cells from the tissue sample after exposure to anantigen of interest or a step of recovering antigen-specific T cellsfrom the tissue sample after exposure to an antigen of interest. In someembodiments, such step of isolating may be carried out by sortingantigen-interacting and/or activated T cells from a tissue sample;likewise, in some embodiments, non-antigen-specific T cells and/orun-activated T cells may be sorted from a tissue sample. Such steps ofthe various embodiments may be carried out by a variety of methodsincluding, but not limited to, (i) peptide-MHC multimer stainingreagents (such as, tetramers, pentamers, or the like), followed bysorting, panning, or otherwise capturing complexes between such reagentsand antigen-specific T cells, (ii) sorting or panning or capturing basedon activation markers, such as CD137, CD154, or others (described morefully below), or (iii) proliferation (and therefore, for example, anincrease in frequency) of antigen-specific T cells overantigen-non-specific T cells. Thus, in some embodiments, said step ofisolating may comprise α step isolating activated T cells; or a step ofseparating activated T cells from the tissue sample. In some of suchembodiments, T cell activation markers, as noted above, may be used tosort, pan or otherwise capture activated T cells, using conventionaltechniques. Generally, a step is taken for obtaining a sample of T cellsfrom a pool of T cells derived from the tissue sample, which pool isenriched in antigen-specific T cells and/or activated T cells. In someembodiments, T cells with an activation marker may be sorted or isolatedusing a binding compound, such as an antibody, which specifically bindsto the activation marker and which can be directly or indirectly labeledin accordance with conventional methods, e.g. FACS, magnetic bead-basedseparation, or like techniques.

In another application of the above embodiment, T cell immunogenicitymay be measured in the following steps: (a) reacting under activationconditions a tissue sample comprising T cells with an antigen or anantigen reagent thereof, (b) sequencing recombined nucleic acidsencoding a T-cell receptor chain or a portion thereof from a sample of Tcells from the tissue sample exposed to antigen or antigen reagentsthereof to provide sequence reads from which clonotypes are determined;(c) isolating activated T cells from the tissue sample; (d) sequencingrecombined nucleic acids encoding a T-cell receptor chain or a portionthereof from a sample of the activated T cells isolated from the tissuesample to provide sequence reads from which clonotypes are determined;and (e) quantifying immunogenicity of the antigen as a function ofincreases in frequencies of clonotypes in the sample of isolated T cellsexposed to antigen with respect to frequencies of the same clonotypes inthe tissue sample prior to said step of isolating. Exemplary functionsof increases in frequencies of clonotypes include an average ofincreases among the isolated antigen-specific T cells; another exemplaryfunction of increases includes an average distance of data points ofclonotypes registering increases in frequency from the diagonal in plotssuch as those of FIG. 17, 18 or 19 . Still another measure of T cellimmunogenicity includes any of several similarity measures of aclonotype profile of T cells of the exposed tissue sample prior toisolating and a clonotype profile of T cells of a sample of T cellsisolated (or separated) from the tissue sample, such as described inFaham et al, International Patent Publication No. WO 2013/036459, whichis incorporated herein by reference. In this embodiment, antigens ofparticular interest are therapeutic proteins, such as therapeuticantibodies.

In one aspect, a similarity measure for use with these embodiments ofthe invention is a monotonically varying function that maps (or iscapable of mapping by a simple transformation) at least two sets ofclonotype frequency measurements (e.g. two sequence-based clonotypeprofiles) to the unit interval [0,1]. Simple transformations include,but are not limited to, any linear transformation of dependentvariables, logarithmic transformations, such as y_(ij)=1n(n_(ij)+1)(where n_(ij) is the number of clonotype i in sample j), or the like. Avalue of zero means no similarity between clonotype profiles and a valueof one means two clonotype profiles are statistically identical.

Exemplary similarity measures that may be implemented in theseembodiments are described in Legendre and Legendre, Numerical Ecology(Elsevier, 1998); Magurran, Measurement of Biological Diversity(Wiley-Blackwell, 2003); Wolda, Occologia (Berl), 50: 296-302 (1981);and like references, which are incorporated by reference. Suchsimilarity measures include, but are not limited to, Czekanowski'sindex, Dice's coefficient, Horn's information theory index, Canberrametric, Morisita's index, Kaczynski's similarity index, Sorensen'sindex, Jacquard's index, Bray-Curtis index, and the like. In one aspect,similarity measures are similarity metrics; or in other words, thesimilarity measures employed have properties of a distance measure, suchas, (i) the value of the measure is always non-negative, (ii) themeasure is zero if and only if the clonotype profile measurements areidentical, (iii) the value of the measure is invariant with respect tothe ordering of the clonotype profile measurements (sometimes expressedas d(x,y)=d(y,x)), (iv) the triangle inequality holds with respect tothree different clonotype profile measurements. In another aspect,similarity measures may be correlation coefficients (subject to a simpletransformation, e.g. taking its absolute value, squaring its value, orthe like, so that its value is restricted to the unit interval).Exemplary correlation coefficients include, but are not limited to,Pearson product-moment correlation coefficient and rank correlations,such as Spearman's rank correlation coefficient, Kendall's tau rankcorrelation coefficient, and the like. In one embodiment a Morisita-Hornindex (C₁₂) (including Morisita-Horn index with a logarithmictransformation), as disclosed in Wolda (cited above), is employed withthe embodiments.

Another embodiment for identifying pairs of immune receptor chains ofantigen-specific T cells is illustrated in FIG. 12D, where T cellcontaining reaction mixtures are exposed to a single antigen. Similarlyto the embodiment of FIG. 12B, a tissue sample is partitioned intosubsets (152) from 1 to K and a portion of the subsets may be selectedfor analysis. Ranges in the values of K and the portion selected may bethe same as for the embodiment of FIG. 12B. In one embodiment, as above,the partitions may be aliquots of the tissue sample, in whichapproximately equal amounts of tissue sample are provided to eachsubset, for example, by distributing equal amounts of tissue sample toeach of K reaction mixtures, which may be contained by vessels orreactors, such as wells in a multi-well plate. Tissue samples may alsobe distributed to a plurality of K separate chambers of a microfluidicsdevice in connection with this and/or the embodiments described above. Tcells of each subset are exposed to antigen (179) after which reactionmixtures in the K vessels are incubated for a time (for example, apredetermined interval) sufficient for T cells to respond to, orinteract with, the antigen, either directly or in a processed form (forexample, as an antigen reagent). Such response may include forming astable complex with antigen or a processed form thereof, or may includethe development and/or expression of activation markers by T cells, ormay include proliferation by T cells specific for the antigen.Antigen-interacting or antigen-responding T cells are then selected(180) and isolated (for example, sorted) from each of the K chambers,after which recombined nucleic acids encoding predetermined portions ofone or both TCR chains are sequenced to provide sequence reads fromwhich clonotypes and clonotype profiles (for example, 154 and 156) areformed. As above with the embodiment of FIG. 12B, once such profiles areobtained they are examined to identify pairs of first and secondnucleotide sequences that either occur in a subset together or are bothabsent from a subset. By way of example, the members of pair (181)appear in lists (184) of subset 2 and in lists (186) of subset K−1, butneither member of the pair appears in lists (182) or (188) of subsets 1and K, either alone or together. As above, this reflects the presence orabsence of a particular lymphocyte, which in this illustration is insubsets 2 and K−1, but is absent from subsets 1 and K. Such a patternconfirms that the members of pair (181) go together and correspond tothe chains of a functional immune receptor that is specific for antigen,Ag₁ (179).

In some embodiments, the above method of determining receptors ofantigen-specific T cells in a tissue sample may comprise the followingsteps: (a) partitioning a tissue sample containing T cells into aplurality of subsets; (b) exposing the T cells of each of a portion ofsubsets to an antigen so that T cells specific for the antigen areactivated; (c) isolating the activated T cells of each subset of theportion; (d) sequencing recombined nucleic acids encoding T-cellreceptor a chains in each subset of the portion to provide sequencereads from which a chain clonotypes are determined; (e) sequencingrecombined nucleic acids encoding T-cell receptor beta chains in eachsubset of the portion to provide sequence reads from which beta chainclonotypes are determined; and (f) identifying as antigen-specific Tcell receptors with those pairs of alpha chain clonotypes and beta chainclonotypes that for every subset of the portion (i) either both the achain clonotype and beta chain clonotype are present in a subset orneither are present in a subset, and (ii) both the a chain clonotype andbeta chain clonotype are present in at least one subset of the portionand the a chain clonotype and beta chain clonotype are not present in atleast one subset of the portion.

Alternatively, in some embodiments, the above method of determiningreceptors of antigen-specific T cells in a tissue sample may comprisethe following steps: (a) forming a plurality of subsets from a tissuesample containing T cells; (b) reacting under activation conditions theT cells of each subset to an antigen; (c) isolating the antigen-specificT cells of each subset; (d) sequencing recombined nucleic acids encodingT-cell receptor a chains in each subset to provide sequence reads fromwhich a chain clonotypes are determined; (e) sequencing recombinednucleic acids encoding T-cell receptor beta chains in each subset toprovide sequence reads from which beta chain clonotypes are determined;(d) identifying as antigen-specific T cell receptors with those pairs ofa chain clonotypes and beta chain clonotypes that for every subset (i)either both the alpha chain clonotype and beta chain clonotype arepresent in a subset or neither are present in a subset, and (ii) boththe a chain clonotype and beta chain clonotype are present in at leastone subset and the a chain clonotype and beta chain clonotype are notpresent in at least one subset. In some of these latter embodiments, theplurality of subsets formed may correspond to a portion of the pluralityinto which a tissue sample is partitioned in the former embodiments. Insome embodiments, the step of forming a plurality of subsets maycomprise aliquoting portions of a tissue sample into separate reactionvessels. In some embodiments, such portions are equal portions.

Another embodiment for identifying clonotypes of antigen-specific Tcells is illustrated in FIGS. 16A-16B, where a plurality of antigens(500) is exposed to T cells in a plurality of different reactionmixtures. In one aspect, this embodiment permits the identification ofantigen-specific T cells from scarce tissue samples, such as from acancer patient whose tissue sample will be used to identify clonotypesfor immune monitoring (e.g. minimal residual disease (MRD) analysis), toproduce a patient-specific immunotherapeutic reagent using cancerantigen-specific T cells, or the like. Subsets (or reaction mixtures)(502) from 1 to K (shown in FIGS. 16A-16B as 1-8) are formed from atissue sample.

The number of different antigens employed may vary widely and in someembodiments the number depends on the nature of the antigens. Forprotein antigens, in some embodiments, a plurality (a number equal to orgreater than 2) of protein antigens may be employed; in furtherembodiments, a plurality of protein antigens may be in the range of from2 to 100 protein antigens; in other embodiments, a plurality may be inthe range of from 2 to 50 protein antigens; in other embodiments, aplurality may be in the range of from 2 to 10 protein antigens; in stillother embodiments, a plurality may be in the range of from 2 to 1000protein antigens. In some embodiments, a plurality of protein antigensmay be in the range of from 10 to 50 protein antigens; in otherembodiments, a plurality may be in the range of from 10 to 100 proteinantigens; in other embodiments, a plurality may be in the range of from10 to 1000 protein antigens; in still other embodiments, a plurality maybe in the range of from 10 to 500 protein antigens; in still otherembodiments, a plurality may be in the range of from 500 to 1000 proteinantigens.

As illustrated in FIGS. 16A and 16B, each antigen of plurality (500) isexposed to (or presented to) T cells of a subplurality of reactionmixtures (502) less than the total plurality of K reaction mixtures (inthis illustration, subpluralities are each 4). Selections of the subsetsof reaction mixtures into which antigens are placed are predeterminedfor each antigen. In some embodiments, each antigen is applied orexposed to a unique subplurality of subsets. That is, the selection ofsubsets making up a subplurality corresponding to a particular antigenis unique to that antigen. The size of the subpluralities may be thesame or different for each antigen; but in some embodiments, the size ofthe subpluralities (i.e. the number of subsets in each) are the same foreach antigen (which is equal to 4 in FIGS. 16A-16B). In someembodiments, subpluralities of subsets correspond to a differentcombination of subsets out of the plurality (in this case 8), asmentioned above. Thus, for some embodiments, the number of possiblesubsets is the same as the number of different combinations of R subsetsselected from the total number of subsets, K. (For example, for R=4 andK=8, the number of different combinations is K!/(R!(K-R)!).

A selection of different combinations (or subpluralities) for antigens(500) is indicated by matrix (506) of +'s and −'s which indicate whichantigen is exposed to T cells of which subsets. As mentioned above, theselection of subsets into which an antigen is applied (or exposed) ispredetermined; thus, for example, antigen Ag₁ is applied to subpluralityof subsets 1, 4, 5 and 7. A subplurality of subsets which are exposed toantigen may vary between 2 and K−1; however, in some embodiments, thesize of the subplurality is an integer equal to or closest to K/2. Asabove, after exposure to antigen and optional incubation,antigen-specific T cells are selected (504) (e.g. based on interactionwith an antigen in the reaction mixture) and clonotype profiles aregenerated for recombined nucleic acids encoding a selected TCR chain ora portion thereof (as illustrated for subset 1), which permits itscorresponding T cell to be identified and/or isolated. Prior toexposure, a sample of T cells may be taken from the tissue samplesubsets (for example, 510). Recombined nucleic acids encoding clonotypesof the same TCR segment are sequenced both in sample (510) and in sample(511) to produce sequence reads (514) and (515) from which clonotypesand clonotype frequencies are determined. Frequencies of clonotypes thatincrease in the selected pools of T cells (illustrated as lists (520) inFIG. 16B) correspond to T cells specific for antigens (for example, Ag1or Ag4 in reaction mixture 1).

An antigen-specific clonotype may be identified by observing a clonotypethat increases in frequency in every reaction mixture of a givenantigen. For example, in FIGS. 16A-16B, the same clonotype (518 a, 518b, 518 c and 518 d) is observed to have increased in frequency withinreaction mixtures 1, 4, 5 and 7 which corresponds to the uniquesubplurality of subsets into which antigen 1 was added, but not to haveincreased in the other reaction mixtures where antigen 1 was absent;therefore, clonotype (518) identifies a T cell with a TCR specific forantigen 1. Likewise, the same clonotype (522 a, 522 b, 522 c and 522 d)is observed to have increased in frequency within reaction mixtures 1,2, 3 and 8, which corresponds to the unique subplurality of subsets intowhich antigen 4 was added, but not to have increased in frequency in theother reaction mixtures where antigen 4 was absent; therefore, clonotype(522) identifies a T cell with a TCR specific for antigen 4. Since eachantigen is exposed to T cells in a unique subplurality of reactionmixtures (or subsets), whenever the same clonotype is observed in eachreaction mixture of the unique subplurality, then the clonotypecorresponds to a TCR specific for the antigen corresponding to thesubplurality.

In one aspect, the above embodiments of the invention for determiningclonotypes of antigen-specific T cells in a tissue sample may be carriedout with the following steps: (a) forming a plurality of subsets from atissue sample containing T cells; (b) exposing under interactionconditions T cells in a subplurality of subsets to one or more antigensso that T cells specific for any of the one or more antigens are capableof interacting therewith, and wherein each different antigen is exposedto T cells in a different subplurality; (c) enriching theantigen-interacting T cells of each subset of a subplurality; (d)sequencing recombined nucleic acids encoding a T-cell receptor chain ora portion thereof from said enriched T cells in each subset of thesubplurality to provide sequence reads from which clonotypes aredetermined; (e) sequencing recombined nucleic acids encoding T-cellreceptor chain or a portion thereof from said T cells in each subset ofthe subplurality prior to said step of enriching or from non-enriched Tcells in each subset of the subplurality to provide sequence reads fromwhich clonotypes are determined; and (f) identifying a clonotype of a Tcell specific for an antigen of the one or more antigens as a clonotypewhose frequency increases in each subset of a subplurality correspondingto the antigen and does not increase in subsets outside of suchsubplurality. That is, in some embodiments, such clonotypes areidentified by observing the clonotypes in all reaction mixtures thatincrease in frequency (520 in FIG. 16B) and identifying clonotypes thatappear in each of the subsets of the subplurality corresponding to agiven antigen and that is absent in all of the other subsets. In otherwords, a clonotype of a T cell specific for an antigen increases infrequency only in the subsets or reaction mixtures to which the antigenwas added and not in the others. In some embodiments, a clonotype of anantigen-specific T cell may be identified whenever the frequency suchclonotype increases in substantially every subset of a subpluralitycorresponding to the antigen and does not increase in substantiallyevery other subset (every subset not part of the subplurality).

For clarity, FIG. 15 illustrates the process of selecting subpluralitiesof a plurality of subset in accordance with some embodiments of theinvention. Tissue sample (400) is separated into a plurality of subsets(402), for example, 10 as shown in FIG. 15 . Tissue sample (400) mayalso be aliquoted into a plurality of subsets, or a plurality of subsetsmay be formed from it, which may or may not use the entire amount oftissue sample (400). A subplurality of plurality (402) is a selection offrom two to nine subsets of plurality (402). In some embodiments,several subpluralities are selected that each have the same number ofsubsets, such as illustrated in FIG. 15 , where each subpluralityconsists of five subsets. In some embodiments of the invention, adifferent antigen is exposed to T cells in subsets of a differentsubplurality. Thus, for example, subplurality 1 may be exposed toantigen 1, subplurality 2 exposed to antigen 2, and so forth.Consequently, in FIG. 15 , subset 1 is exposed to antigen 1, antigen 3and antigen 4; likewise, subset 2 is exposed to antigen 1 and antigen 2;and so forth.

IV. Identification of Antigen-Specific T Cell Clonotypes Using SingleCell Techniques

In some embodiments, antigen-specific T cell clonotypes may beidentified using single cell techniques, such as disclosed in Faham andWillis, U.S. Pat. Nos. 8,236,503 and 8,507,205, which are incorporatedherein by reference. In one aspect, the step of selecting T cells thatinteract with antigen is carried out by disposing T cells exposed toantigen(s) into reactors so that a substantial fraction of reactorscontain a single T cell and a single labeled antigen reagent, usuallybound to a TCR of the T cell. An objective of these embodiments of theinvention is to carry out a polymerase cycling assembly (PCA) reaction(also sometimes referred to as a “linking PCR”) on individual cells inthe reactors to link their recombined nucleic acid sequences (e.g.,encoding a portion of a TCR) to a sequence tag that is associated with,or labels, an antigen reagent present in the reactor with the single Tcell. The products of such linking are referred to herein as “fusionproducts.” After their generation, fusion products can be sequenced toidentify both the clonotype of the TCR and the sequence tag which, inturn, identifies the antigen reagent. FIG. 12E gives an overview on oneembodiment of the invention. Lymphoid cells (1010) (shown combined withantigen reagents (1000)) each have a distinct identifying nucleic acid(1012), which in the figure are exemplified (without any intention ofbeing limiting) as messenger RNAs (mRNAs) (1012), which in the threecells illustrated in the figure are labeled “C₁”, “C₂”, and “C₃”, toindicate that they are three different recombined nucleic acids uniqueto each cell, respectively. These recombined nucleic acids encode TCRs(for example, 1001) expressed on the surface of the respective T cells.As mentioned above, T cells (1010) are shown combined with antigenreagents (1000), which may be conventional multimers, such as tetramers,which are labeled with sequence tags (for example, 1003) that identifythe MHC and peptide portions of the antigen reagent (for example, asshown enclosed in dashed box 1004). Antigen reagent (1000) isexemplified with a conventional structure comprising a frameworkcomponent (1002), such as a streptavidin molecule; MHC linking moieties(such as, biotinylated peptides (for example, 1006)); and MHC-peptidecomplexes (1004).

Antigen reagent (1000) may also include sequence tag labels (such as,1003), which may be produced as taught by Kwong et al, U.S. Pat. No.8,394,590, which is incorporated herein by reference. The MHC andpeptide portion determines the specificity of the reagent for a TCR andvice versa. Antigen reagents (1000) are produced so that substantiallyevery different antigen reagent (e.g. every different multimer) has adifferent sequence tag. In some embodiments, sequence tags andMHC-peptide portions are selected so that with the knowledge of a tag'ssequence, the identity of the MHC-peptide portion can be uniquelydetermined. That is, for example, there is a one-to-one correspondencebetween a sequence tag and an MHC-peptide complex, so that (forexample), a sequence tag “X” indicates the presence of recombinednucleic acid “C₁”, a sequence tag “Y” indicates the presence ofrecombined nucleic acid “C₂”, and a sequence tag “Z” indicates thepresence of recombined nucleic acid “C₃”. Antigen reagents (1000) arecombined (1008) with T cells (1010) in a reaction mixture and areincubated under antigen-interaction conditions which permit theformation of T cell-reagent complexes whenever a TCR is specific for anantigen reagent. After such incubation, cells are disposed (1016) insingle cell reactors, which may vary widely and may include, but not belimited to, plates with arrays of nanoliter-volume wells, microfluidicdevices, and the like, as described more fully below. In someembodiments, single cell reactors are aqueous micelles in an emulsion,such as illustrated (1017) in FIG. 12E, where a substantial fraction ofmicelles in the emulsion contain a single T cell together with a singleantigen reagent. In one aspect, single-cell emulsion (126) is generatedusing a microfluidic emulsion generator, such as disclosed by Zeng etal, Anal. Chem., 82: 3183-3190 (2010), or the like.

Reactors (1018) contain a PCA reaction mixture that, for example, maycomprise α nucleic acid polymerase, outer primers and linking primers(described more fully below), nucleoside triphosphates, a buffersolution, and the like. In some embodiments, a PCA reaction mixture mayalso include one or more cell lysing reagents, to give the foregoingreagents access to intracellular recombined nucleic acids, such asmRNAs. For each reactor (1018) containing a cell, PCA reaction (1020)generates fusion products (1022) that may comprise one or more pairs ofsequences, such that one member of the pair is a sequence tag and theother member is a predetermined recombined nucleic acid. In otherembodiments, fusion products may comprise triplets of sequences, orhigher order concatenations, for example, as taught by Faham and Willis,U.S. Pat. No. 8,507,205. In some embodiments of the method of theinvention, a single kind of fusion product may be generated for eachcell (or per reactor) or a plurality of different kinds of fusionproducts may be generated for each cell (or per reactor). Such pluralitymay be at least 2, or it may be in the range of from 2 to 500, or from 2to 200, or from 2 to 100, or from 2 to 20. In one embodiment, suchplurality may be in the range of from 2 to 10. In some embodiments, suchplurality is two.

After completion of PCA reaction (1020), emulsion (1017) is broken andfusion products (1026) are isolated (1024). Fusion products (1026) arerepresented in FIG. 12E as conjugates of sequence tags (X, Y or Z) andrecombined nucleic acids (e.g. clonotypes) (C₁, C₂ and C₃). A variety ofconventional methods may be used to isolate fusion products (1026) fromthe reaction mixture, including, but not limited to, columnchromatography, ethanol precipitation, affinity purification after useof biotinylated primers, gel electrophoresis, and the like. As part ofPCA reaction (1020) or after isolation (1024), additional sequences maybe added to fusion products (1026) as necessary for sequencing (1028).Sequencing may be carried out using a conventional high-throughputinstrument, e.g. Genome Analyzer IIx (Illumina, Inc., San Diego), or thelike.

Polymerase cycling assembly (PCA) reactions permit a plurality ofnucleic acid fragments to be fused together to form a single fusionproduct in one or more cycles of fragment annealing and polymeraseextension, e.g. Xiong et al, FEBS Micro biol. Rev., 32: 522-540 (2008).PCA reactions come in many formats. In one format of interest, PCAfollows a plurality of polymerase chain reactions (PCRs) taking place ina common reaction volume, wherein each component PCR includes at leastone linking primer that permits strands from the resulting amplicon toanneal to strands from another amplicon in the reaction and to beextended to form a fusion product or a precursor of a fusion product.PCA in its various formats (and under various alternative names) is awell-known method for fragment assembly and gene synthesis, severalforms of which are disclosed in the following references: Yon et al,Nucleic Acids Research, 17: 4895 (1989); Chen et al, J. Am. Chem. Soc.,116: 8799-8800 (1994); Stemmer et al, Gene, 164: 49-53 (1995); Hoover etal, Nucleic Acids Research, 30: e43 (2002); Xiong et al, BiotechnologyAdvances, 26: 121-134 (2008); Xiong et al, FEBS Microbiol. Rev., 32:522-540 (2008); and the like.

An exemplary (but not limiting) PCA format useful in the presentembodiments is described in FIG. 12F, which illustrates a PCA scheme forjoining two separate fragments A′ (1208) and B′ (1210) into a singlefusion product (1222). Fragment A′ (1208) is amplified with primers(1200) and (1202) and fragment B′ (1210) is amplified with primers(1206) and (1204) in the same PCR mixture. Primers (1200) and (1206) are“outer” primers of the PCA reaction and primers (1202) and (1204) arethe “inner” primers of the PCA reaction. Inner primers (1202) and (1204)each have a tail (1203 and 1205, respectively) that are notcomplementary to A′ or B′ (or adjacent sequences if A′ and B′ aresegments imbedded in a longer sequence). Tails (1203) and (1205) arecomplementary to one another. Generally, such inner primer tails areselected for selective hybridization to its corresponding inner primer(and not elsewhere); but otherwise such tails may vary widely in lengthand sequence. In one aspect, such tails have a length in the range offrom 8 to 30 nucleotides; or a length in the range of from 14 to 24nucleotides. As the PCRs progress (1212), product fragments A (1215) andB (1217) are produced that incorporate tails (1203) and (1205) into endregions (1214) and (1216), respectively. During the PCRs productfragments A (1215) and B (1217) will denature and some of the “upper”strands (1215 a) of A anneal (1218) to lower strands (1217 b) of B andthe 3′ ends are extended (1219) to form (1220) fusion product A-B(1222). Fusion product A-B (1222) may be further amplified by an excessof outer primers (1200) and (1206). In some embodiments, the region offusion product (1222) formed from tails (1203) and (1205) may includeone or more primer binding sites for use in later analysis, such ashigh-throughput sequencing. Typically, in PCA reactions theconcentrations of outer primers are greater than the concentrations ofinner primers so that amplification of the fusion product continuesafter initial formation. For example, in one embodiment for fusing twotarget nucleic acids outer primer concentration may be from about 10 to100 times that of the inner primers, e.g. 1 μM for outer primers and0.01 μM for inner primers. Otherwise, a PCA reaction may comprise thecomponents of a PCR.

Single Cell Analysis

As mentioned above, in some embodiments of the invention, cells from apopulation are disposed in reactors each containing a single cell. Thismay be accomplished by a variety of large-scale single-cell reactorplatforms known in the art, e.g. U.S. Patent Application PublicationNos. 2010/0255471, 2010/0285975, 2010/0021984, 2010/0173394, and2009/0181859; and International Patent Publication No. WO 2009/145925;Novak et al, Angew. Chem. Int. Ed., 50: 390-395 (2011); Chen et al,Biomed Microdevices, 11: 1223-1231 (2009); and the like, which areincorporated herein by reference.

In one aspect, cells are disposed in wells of a microwell array wherereactions, such as PCA reactions, take place; in another aspect, cellsare disposed in micelles of a water-in-oil emulsion, where micellesserve as reactors. Micelle reactors generated by microfluidics devices,e.g. Mathies et al (cited above) or Edd et al (cited above), are ofparticular interest because uniform-sized micelles may be generated withlower shear and stress on cells than in bulk emulsification processes.Compositions and techniques for emulsifications, including carrying outamplification reactions, such as PCRs, in micelles is found in thefollowing references, which are incorporated by reference: Becher,“Emulsions: Theory and Practice,” (Oxford University Press, 2001);Griffiths and Tawfik, U.S. Pat. Nos. 6,489,103; 7,842,457; 8,012,690;8,048,627; Tawfik and Griffiths, Nature Biotechnology, 16: 652-656(1998); Nakano et al, J. Biotechnology, 102: 117-124 (2003); Dressman etal, Proc. Natl. Acad. Sci., 100: 8817-8822 (2003); Diehl et al, NatureMethods, 3: 551-559 (2006); Williams et al, Nature Methods, 3: 545-550(2006); Zeng et al, Analytical Chemistry, 82(8): 3183-3190 (2010);Micellula DNA Emulsion & Purification Kit instructions (EUR_(x), Gdansk,Poland, 2011); and the like.

In one embodiment, the mixture of homogeneous sequence tags (e.g. beads)and reaction mixture is added dropwise into a spinning mixture ofbiocompatible oil (e.g., light mineral oil, Sigma) and allowed toemulsify. In another embodiment, the homogeneous sequence tags andreaction mixture are added dropwise into a cross-flow of biocompatibleoil. The oil used may be supplemented with one or more biocompatibleemulsion stabilizers. These emulsion stabilizers may include Atlox 4912,Span 80, and other recognized and commercially available suitablestabilizers. In some embodiments, the emulsion is heat stable to allowthermal cycling, e.g., to at least 94° C., at least 95° C., or at least96° C. Preferably, the droplets formed range in size from about 5microns to about 500 microns, more preferably from about 10 microns toabout 350 microns, even more preferably from about 50 to 250 microns,and most preferably from about 100 microns to about 200 microns.Advantageously, cross-flow fluid mixing allows for control of thedroplet formation, and uniformity of droplet size.

In some embodiments, micelles are produced having a uniform distributionof volumes so that reagents available in such reactors result insimilarly amplified target nucleic acids and sequence tags. That is,widely varying reactor volumes, e.g. micelle volumes, may lead toamplification failures and/or widely varying degrees of amplification.Such failures and variation would preclude or increase the difficulty ofmaking quantitative comparisons of target nucleic acids in individualcells of a population, e.g. differences in gene expression. In oneaspect, micelles are produced that have a distribution of volumes with acoefficient of variation (CV) of thirty percent or less. In someembodiments, micelles have a distribution of volumes with a CV of twentypercent of less.

Cells of a tissue sample and antigen reagent may be suspended in areaction mixture prior to disposition into reactors. In one aspect, areaction mixture is a PCA reaction mixture and is substantially the sameas a PCR reaction mixture with at least one pair of inner (or linking)primers and at least one pair of outer primers. In some embodiments, astep of lysing cells may be accomplished by heating cells to atemperature of 95° C. or above in the presence of a nonionic detergent,e.g. 0.1% Triton X-100 or Tween-20, for a period prior to carrying outan amplification reaction. In one embodiment, such period of elevatedtemperature may be from 10-20 minutes. Alternatively, a step of lysingcells may be accomplished by one or more cycles of heating and cooling,e.g. 96° C. for 15 min followed by 10° C. for 10 min, in the presence ofa nonionic detergent, e.g. 0.1% Triton X-100 or Tween-20. Guidance forcarrying out a lysing step is disclosed in Brown et al, J. R. Soc.Interface 5: S131-S138 (2008).

Clearly many microfluidics device configurations may be employed togenerate micelles containing single cells, e.g. Zagoni et al, Chapter 2,Methods of Cell Biology, 102: 25-48 (2011); Bronzes, Chapter 10, Methodsof Cell Biology, 102: 105-139 (2011); Wiklund et al, Chapter 14, Methodsof Cell Biology, 102: 177-196 (2011); Le Gac et al, Chapter 7, Methodsof Molecular Biology, 853: 65-82 (2012); and the like.

In some embodiments, this aspect of the invention for determiningantigen-specific T cells may be implemented with the following steps:(a) exposing under interaction conditions a tissue sample containing Tcells to antigen reagents labeled with sequence tags; (b) disposing inmultiple reactors single T cells specifically bound to at least oneantigen reagent, each reactor containing a polymerase cycling assembly(PCA) reaction mixture comprising a pair of outer primers and one ormore pairs of linking primers, at least one pair of such outer andlinking primers being specific for a recombined nucleic acid encoding asegment of a TCR chain of the T cell and one or more pairs of such outerand linking primers being specific for a sequence tag attached to theantigen reagent; (c) performing a PCA reaction in the reactors to formfusion products comprising said recombined nucleic acids and saidsequence tag; (d) spatially isolating individual molecules of fusionproducts from the reactors; (e) sequencing the spatially isolated fusionproducts from the reactors to generate sequence reads from which pairsof clonotypes and sequence tags are determined; and (f) identifyingantigen-specificity of T cells by their clonotype and sequence tagpairs. In some embodiments, the reactors are aqueous micelles of awater-in-oil emulsion. In some embodiments, aqueous micelles aregenerated by a microfluidics device. In some embodiments, the reactorsare nanoliter wells in a planar substrate. In some embodiments, afurther step of lysing the single T cells in the reactors is carried outprior to performing the PCA reaction.

V. Antigen-Specific TCRs

In some embodiments, the present disclosure provides antigen-specificTCRs (e.g., antigen-specific TCR alpha and/or TCR beta chains). In someembodiments, such TCRs are specific for an epitope on the oncoproteinWT1. WT1 is a multifunctional zinc finger transcription factor that wasoriginally identified as a tumor suppressor gene associated with Wilms'tumor, a nephroblastoma. However, WT1 is complex and more recently hasbeen appreciated as an oncogene. Aberrant or overexpressed WT1 has beenshown in a variety of cancers including Wilms' tumors, chronicmyelocytic leukemia, multiple myeloma, acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), myelodysplastic syndrome,mesothelioma, ovarian cancer, breast cancer, prostate cancer,gastrointestinal cancers, lung cancer, colon cancer, thyroid cancer,head and neck cancer, glioblastoma, and sarcoma, among others. Inparticular, clinical and experimental data indicate that WT1 is apotentially useful target for treatment of leukemias.

The WT1 epitope may be any WT1 antigen. In particular embodiments, theWT1 antigen is a WT1 peptide comprising, consisting essentially of, orconsisting of SEQ ID NO: 97. Thus, in one aspect, the present disclosureprovides TCRs that bind to an epitope of the WT protein. The TCRsprovided herein, in some embodiments, recognize WT1 epitopes that areHLA-A2 restricted. In humans, approximately 50% of the populationexpresses the MHC class I molecule HLA-A2. HLA-A*02 alleles includeHLA-A*0201, *0202, *0203, *0206, and *0207. The TCRs provided hereinbind strongly to the WT1 antigen (SEQ ID NO: 97) when loaded byHLA-A*0201. In some embodiments, the TCRs of the invention bind to theWT1 protein antigen when loaded by other HLAs, for example, HLA-A*0203and HLA-A*0206.

The TCRs provided may bind with different strengths to the WT1 antigen.In some embodiments, the TCRs bind strongly to the WT1 antigen andexhibit potent activity. In some embodiments, the TCRs bind strongly tothe WT1 antigen but do not exhibit potent activity. In some embodiments,the TCRs do not bind strongly to the WT1 protein or bind with lower ormoderate on/off kinetics, but exhibit highly potent activity. Forexample, in some embodiments, the TCRs provided herein exhibit highfunctional avidity and/or high cytolytic activity but do not exhibitlong t_(1/2) or slow off rate kinetics.

In some embodiments, the TCRs of the invention bind a WT1/HLA-A2 complexand elicit functional T cell activity. As referred to herein, the EC₅₀refers to the effective concentration of antigen that is loaded ontoantigen-presenting cells to elicit a half maximal response of antigenspecific activation or cytolysis. In some embodiments, the TCRs of theinvention have an EC₅₀ for activation of less than about 1 μM, less thanabout 500 nM, less than about 100 nM, less than about 75 nM, less thanabout 50 nM, less than about 25 nM, less than about 20 nM, less thanabout 15 nM, less than about 10 nM, less than about 5 nM, less thanabout 1 nM, less than about 500 μM, less than about 100 μM, or less thanabout 1 μM. In some embodiments, the TCRs of the invention have an EC₅₀for cytolysis of less than about 1 μM, less than about 500 nM, less thanabout 100 nM, less than about 75 nM, less than about 50 nM, less thanabout 25 nM, less than about 20 nM, less than about 15 nM, less thanabout 10 nM, less than about 5 nM, less than about 1 nM, less than about500 μM, less than about 100 μM, or less than about 1 μM. In someembodiments, the EC₅₀ for the TCRs provided herein is the concentrationof the antigen loaded onto antigen presenting cells (such as T2 cells)that induces 50% of the maximum response.

In some embodiments, the present disclosure provides recombinant TCRswhich specifically bind to an epitope of the WT1 protein and have highfunctional avidity. In some embodiments, the epitope comprises an aminoacid sequence set forth in SEQ ID NO: 97. In some embodiments, the highfunctional avidity is measured by T cell activation and/or Tcell-mediated cytolysis. In some embodiments, the high functionalavidity is measured by the EC₅₀ for T cell activation and/or Tcell-mediated cytolysis. Thus, in some embodiments, the presentdisclosure provides TCRs that specifically bind to an epitope of the WT1protein and have an EC₅₀ for activation and/or cytolysis of less thanabout 1 μM, less than about 500 nM, less than about 100 nM, less thanabout 75 nM, less than about 50 nM, less than about 25 nM, less thanabout 20 nM, less than about 15 nM, less than about 10 nM, less thanabout 5 nM, less than about 1 nM, less than about 500 μM, less thanabout 100 μM, or less than about 1 μM.

TCRs may be provided as individual chains, portions thereof, or ascomplete molecules comprising an alpha and a beta chain. In someembodiments, each of the TCR alpha and beta chains comprise threecomplementarity determining regions (CDRs), CDR1, CDR2, and CDR3. Theseregions determine the specificity of the TCR. The CDR1 and CDR2 regionsare each found in the variable domain (V). The more diverse CDR3 regionof a T cell includes a portion of V, the diverse (D) heavy chains andjoint (J) chains and is therefore critical in imparting both diversityof the TCR repertoire as a whole and the antigen specificity of each Tcell within the repertoire. CDRs of the alpha and beta chains aregenerally defined using the nomenclature system used in theImmunogenetics (IMGT) database, but may also be longer or shorter thanin the IMGT database system. For example, in some embodiments, the betachain CDR3 may include the amino acid sequence for the beta chain CDR3provided herein and additional amino acids C-terminal and/or N-terminalto the terminal amino acids of the CDR sequence provided. CDRs may alsobe defined by other nomenclature systems known in the art, such as, forexample, the Garcia system (Garcia et al. (1999), Ann. Rev. Immunol 17,369, incorporated herein by reference in its entirety).

TCRs may be defined by amino acid sequence and/or nucleic acid sequence.In some embodiments, the nucleic acid and amino acid sequencesencompassed in the present disclosure have at least 75%, at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to the particular sequences disclosedherein. In some embodiments, amino acid sequences have one or moresubstitution, deletion, or insertion. In some embodiments, amino acidsequences have two or more, three or more, four or more, or five or moresubstitutions, deletions, or insertions. In some embodiments, the aminoacid substitutions are conservative amino acid substitutions.“Conservative amino acid substitutions” is understood by the skilledartisan to mean that codons encoding positively-charged residues (H, K,and R) are substituted with codons encoding positively-charged residues,codons encoding negatively-charged residues (D and E) are substitutedwith codons encoding negatively-charged residues, codons encodingneutral polar residues (C, G, N, Q, S, T, and Y) are substituted withcodons encoding neutral polar residues, and codons encoding neutralnon-polar residues (A, F, I, L, M, P, V, and W) are substituted withcodons encoding neutral non-polar residues. These variations canspontaneously occur, be introduced by random mutagenesis, or can beintroduced by directed mutagenesis. Those changes can be made withoutdestroying the essential characteristics of these polypeptides, whichare to recognize their cognate antigen in the context of an MHC. Theordinarily skilled artisan can readily and routinely screen variantamino acids and/or the nucleic acids encoding them to determine if thesevariations substantially lessen or destroy the ligand binding capacityby methods known in the art.

In some embodiments, the TCR (e.g., a WT1-specific TCR) comprises analpha chain CDR1 having an amino acid sequence with at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1, 9, 17, 25, 33, and 41. In furtherembodiments, the TCR alpha chain CDR1 comprises or consists of an aminoacid sequence selected from the group consisting of SEQ ID NOs: 1, 9,17, 25, 33, and 41. In some embodiments, the TCR comprises an alphachain CDR2 having an amino acid sequence with at least 85%, at least90%, at least 95%, at least 96%, at least 97% at least 98%, or at least99% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2, 10, 18, 26, 34, and 42. In furtherembodiments, the TCR alpha chain CDR2 comprises or consists of an aminoacid sequence selected from the group consisting of SEQ ID NOs: 2, 10,18, 26, 34, and 42. In some embodiments, the TCR comprises an alphachain CDR3 having an amino acid sequence with at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 3, 11, 19, 27, 35, and 43. In furtherembodiments, the TCR alpha chain CDR3 comprises or consists of an aminoacid sequence selected from the group consisting of SEQ ID NO: 3, 11,19, 27, 35, and 43.

In some embodiments, the TCR (e.g., a WT1-specific TCR) comprises a betachain CDR1 having an amino acid sequence with at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, 12, 20, 28, 36, and 44. In furtherembodiments, the TCR beta chain CDR1 comprises or consists of an aminoacid sequence selected from the group consisting of SEQ ID NO: 4, 12,20, 28, 36, and 44. In some embodiments, the TCR comprises beta chainCDR2 having an amino acid sequence with at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 5, 13, 21, 29, 37, and 45. In furtherembodiments, the TCR beta chain CDR2 comprises or consists of an aminoacid sequence selected from the group consisting of SEQ ID NO: 5, 13,21, 29, 37, and 45. In some embodiments, the TCR comprises beta chainCDR3 having an amino acid sequence with at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 6, 14, 22, 30, 38, and 46. In furtherembodiments, the TCR beta chain CDR3 comprises or consists of an aminoacid sequence selected from the group consisting of SEQ ID NO: 6, 14,22, 30, 38, and 46.

In some embodiments, the TCR (e.g., a WT1-specific TCR) comprises analpha chain amino acid sequence with at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NO: 7, 15, 23, 31, 39, and47. In some embodiments, the alpha chain TCR comprises or consists of anamino acid sequence selected from the group consisting of SEQ ID NO: 7,15, 23, 31, 39, and 47. In some embodiments, the beta chain TCRcomprises an amino acid sequence with at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NO: 8, 16, 24, 32, 40, and48. In some embodiments, the beta chain TCR comprises or consists of anamino acid sequence selected from the group consisting of SEQ ID NO: 8,16, 24, 32, 40, and 48.

In some embodiments, the TCR (e.g., a WT1-specific TCR) comprises, as acomplete molecule, paired alpha and beta chains, wherein the alpha chaincomprises an amino acid sequence with at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7 andthe beta chain comprises an amino acid sequence with at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to SEQID NO: 8. In some embodiments, the TCR (e.g., a WT1-specific TCR)comprises, as a complete molecule, paired alpha and beta chains, whereinthe alpha chain comprises or consists of an amino acid sequence of SEQID NO: 7 and the beta chain comprises or consists of an amino acidsequence of SEQ ID NO: 8.

In some embodiments, the TCR (e.g., a WT1-specific TCR) comprises, as acomplete molecule, paired alpha and beta chains, wherein the alpha chaincomprises an amino acid sequence with at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15 andthe beta chain comprises an amino acid sequence with at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to SEQID NO: 16. In some embodiments, the TCR (e.g., a WT1-specific TCR)comprises, as a complete molecule, paired alpha and beta chains, whereinthe alpha chain comprises or consists of an amino acid sequence of SEQID NO: 15 and the beta chain comprises or consists of an amino acidsequence of SEQ ID NO: 16.

In some embodiments, the TCR (e.g., a WT1-specific TCR) comprises, as acomplete molecule, paired alpha and beta chains, wherein the alpha chaincomprises an amino acid sequence with at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23 andthe beta chain comprises an amino acid sequence with at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to SEQID NO: 24. In some embodiments, the TCR (e.g., a WT1-specific TCR)comprises, as a complete molecule, paired alpha and beta chains, whereinthe alpha chain comprises or consists of an amino acid sequence of SEQID NO: 23 and the beta chain comprises or consists of an amino acidsequence of SEQ ID NO: 24.

In some embodiments, the TCR (e.g., a WT1-specific TCR) comprises, as acomplete molecule, paired alpha and beta chains, wherein the alpha chaincomprises an amino acid sequence with at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31 andthe beta chain comprises an amino acid sequence with at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to SEQID NO: 32. In some embodiments, the TCR (e.g., a WT1-specific TCR)comprises, as a complete molecule, paired alpha and beta chains, whereinthe alpha chain comprises or consists of an amino acid sequence of SEQID NO: 31 and the beta chain comprises or consists of an amino acidsequence of SEQ ID NO: 32.

In some embodiments, the TCR (e.g., a WT1-specific TCR) comprises, as acomplete molecule, paired alpha and beta chains, wherein the alpha chaincomprises an amino acid sequence with at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39 andthe beta chain comprises an amino acid sequence with at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to SEQID NO: 40. In some embodiments, the TCR (e.g., a WT1-specific TCR)comprises, as a complete molecule, paired alpha and beta chains, whereinthe alpha chain comprises or consists of an amino acid sequence of SEQID NO: 39 and the beta chain comprises or consists of an amino acidsequence of SEQ ID NO: 40.

In some embodiments, the TCR (e.g., a WT1-specific TCR) comprises, as acomplete molecule, paired alpha and beta chains, wherein the alpha chaincomprises an amino acid sequence with at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 47 andthe beta chain comprises an amino acid sequence with at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to SEQID NO: 48. In some embodiments, the TCR (e.g., a WT1-specific TCR)comprises, as a complete molecule, paired alpha and beta chains, whereinthe alpha chain comprises or consists of an amino acid sequence of SEQID NO: 47 and the beta chain comprises or consists of an amino acidsequence of SEQ ID NO: 48.

With reference to the tables of sequences provided herein, TCRsaccording to the invention may also be referred to as eJH30_WT1C_5 (usedinterchangeably herein with the terms “eJH30_5” and “30_5”).eJH30_WT1C_5 comprises an alpha chain TCR amino acid sequence set forthin SEQ ID NO: 7 and a beta chain TCR amino acid sequence set forth inSEQ ID NO: 8. In some embodiments, a TCR provided herein is referred toas eJH30_WT1C_8 (used interchangeably herein with the terms “eJH30_8”and “30_8”). eJH30_WT1C_8 comprises an alpha chain TCR amino acidsequence set forth in SEQ ID NO: 15 and a beta chain TCR amino acidsequence set forth in SEQ ID NO: 16. In some embodiments, a TCR providedherein is referred to as eJH52_WT1C_13 (used interchangeably herein withthe terms “eJH52_13” and “52_13). eJH52_WT1C_13 comprises an alpha chainTCR amino acid sequence set forth in SEQ ID NO: 23 and a beta chain TCRamino acid sequence set forth in SEQ ID NO: 24. In some embodiments, aTCR provided herein is referred to as eJH64_WT1C_9 (used interchangeablyherein with the terms “eJH64_9” and “64_9”). eJH64_WT1C_9 comprises analpha chain TCR amino acid sequence set forth in SEQ ID NO: 31 and abeta chain TCR amino acid sequence set forth in SEQ ID NO: 32. In someembodiments, a TCR provided herein is referred to as eJH64_WT1C_6 (usedinterchangeably herein with the terms “eJH64_6” and “64_6”).eJH64_WT1C_6 comprises an alpha chain TCR amino acid sequence set forthin SEQ ID NO: 39 and a beta chain TCR amino acid sequence set forth inSEQ ID NO: 40. In some embodiments, a TCR provided herein is referred toas eJH30_WT 1C_7 (used interchangeably herein with the terms “eJH30_7”and “30_7”). eJH30_WT1C_7 comprises an alpha chain TCR amino acidsequence set forth in SEQ ID NO: 47 and a beta chain TCR amino acidsequence set forth in SEQ ID NO: 48.

Advantageously, a TCR as provided herein exhibits high functionalavidity and/or high cytolytic activity in vitro. In other embodiments,the TCR provided herein exhibits moderate functional avidity and/ormoderate cytolytic activity in vitro. In some embodiments, higherfunctional avidity and in vitro activity corresponds with higher in vivopotency. In other embodiments, desirable in vivo potency is associatedwith a particular range of functional avidity and cytolytic activity invitro, such as, for example, moderate avidity and cytolytic in vitroactivity. In some embodiments, the TCRs provided herein exhibit theability to recognize an endogenous WT1 peptide that is presented on thesurface of a cell in the context of endogenously expressed MHCmolecules. In some embodiments, the TCRs provided herein recognize theendogenous WT1 peptides with a greater affinity and/or avidity thanpreviously described TCRs recognizing WT1 peptides.

TCR avidity for a particular antigen determines the value of the TCR fortherapeutic approaches. For example, in cancer patients, treatment byadoptive immunotherapy of cells expressing TCRs with high avidity for acancer antigen can be an effective treatment. TCR avidity is acombination of the affinity that it has for the presented antigen, whichis determined by the on-rate and off-rate the TCR/peptide-MHCinteraction, and the density at which it is displayed on the surface ofthe T cell. Flow cytometry-based technologies exist that allow thediscrimination of T cells that express high affinity and high avidityTCRs due to fast on-rates or slow off-rates in combination with itssurface display density. These technologies can be used in conjunctionwith the described technology to identify high affinity TCRs based onthese parameters.

In some embodiments, the WT1 TCRs provided herein exhibit a tetramerhalf-life of about 30 seconds to about 100 seconds. In some embodiments,the WT1 TCRs provided herein exhibit a tetramer half-life of about 100seconds to about 300 seconds. In some embodiments, the WT1 TCRs providedherein exhibit a tetramer half-life of less than about 100 seconds orabout 100 to about 300 seconds. In some embodiments, the WT1 TCRsprovided herein exhibit a tetramer half-life of about 300 seconds toabout 1200 seconds. In further embodiments, the TCRs exhibit a tetramerhalf-life of about 600 seconds to about 900 seconds. In someembodiments, the TCRs exhibit a tetramer half-life of at least about 30,at least about 50, at least about 100, 300, at least about 400, at leastabout 500, at least about 600, at least about 700, at least about 800,at least about 900, at least about 1000, or more seconds. In someembodiments, the TCRs exhibit a tetramer half-life of about 600, about625, about 650, about 700, about 750, about 800, about 850, about 900 orabout 950 seconds.

In some embodiments, the present disclosure describes nucleic acidsencoding the TCRs or portions thereof (e.g., nucleic acids encoding aWT1-specific TCR, or portions thereof). The nucleic acids, in someembodiments, comprise α nucleic acid sequence having at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to asequence selected from the group consisting of SEQ ID NOs: 49-96. Insome embodiments, the nucleic acids encoding the TCRs described hereinare provided as cDNA molecules. In some embodiments, the nucleic acidsencoding the TCRs described herein are provided as in vitro transcribedRNA (ivtRNA) molecules. In some embodiments, the present disclosureprovides vectors comprising the nucleic acids provided herein. In someembodiments, a vector comprising a nucleic acid encoding a TCR providedherein is used to transduce or transfect a cell, such that the cell willexogenously express the TCR encoded by the nucleic acid. Transducing ortransfecting the cells with nucleic acids provided herein may beachieved with any method known in the art.

In some embodiments, the present disclosure provides a nucleic acidsequence encoding a TCR alpha chain comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 55, 63, 71, 79, 87,and 95 or a sequence having at least 90%, at least 95%, or at least 99%sequence identity to a sequence selected from the group consisting ofSEQ ID NOs: 55, 63, 71, 79, 87, and 95. In some embodiments, the presentdisclosure provides nucleic acid sequence encoding a TCR beta chaincomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NO: 56, 64, 72, 80, 88, and 96, or a sequence having at least90%, at least 95%, or at least 99% sequence identity to a sequenceselected from the group consisting of SEQ ID NOs: 56, 64, 72, 80, 88,and 96.

In some embodiments, the present disclosure provides a first nucleicacid sequence encoding a TCR alpha chain and a second nucleic acidsequence encoding a TCR beta chain, wherein the first nucleic acidsequence is at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 55 and wherein thesecond nucleic acid sequence is at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:56. In some embodiments, the first nucleic acid sequence comprises orconsists of SEQ ID NO: 55 and the second nucleic acid sequence comprisesor consists of SEQ ID NO: 56. In some embodiments, the first nucleicacid sequence is at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99% identical to SEQ ID NO: 63 and the secondnucleic acid sequence is at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to SEQ ID NO: 64. Insome embodiments, the first nucleic acid sequence comprises or consistsof SEQ ID NO: 63 and the second nucleic acid sequence comprises orconsists of SEQ ID NO: 64.

In some embodiments, the present disclosure provides a first nucleicacid sequence encoding a TCR alpha chain and a second nucleic acidsequence encoding a TCR beta chain, wherein the first nucleic acidsequence is at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 71 and wherein thesecond nucleic acid sequence is at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:72. In some embodiments, the first nucleic acid sequence comprises orconsists of SEQ ID NO: 71 and the second nucleic acid sequence comprisesor consists of SEQ ID NO: 72. In some embodiments, the first nucleicacid sequence is at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99% identical to SEQ ID NO: 79 and the secondnucleic acid sequence is at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to SEQ ID NO: 80. Insome embodiments, the first nucleic acid sequence comprises or consistsof SEQ ID NO: 79 and the second nucleic acid sequence comprises orconsists of SEQ ID NO: 80.

In some embodiments, the present disclosure provides a first nucleicacid sequence encoding a TCR alpha chain and a second nucleic acidsequence encoding a TCR beta chain, wherein the first nucleic acidsequence is at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 87 and wherein thesecond nucleic acid sequence is at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:88. In some embodiments, the first nucleic acid sequence comprises orconsists of SEQ ID NO: 87 and the second nucleic acid sequence comprisesor consists of SEQ ID NO: 88. In some embodiments, the first nucleicacid sequence is at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99% identical to SEQ ID NO: 95 and the secondnucleic acid sequence is at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to SEQ ID NO: 96. Insome embodiments, the first nucleic acid sequence comprises or consistsof SEQ ID NO: 95 and the second nucleic acid sequence comprises orconsists of SEQ ID NO: 96.

The present disclose further encompasses TCRs with enhanced orpreferential pairing of chains. A limitation in the use of T celltherapies using cells expressing exogenous TCRs has been the mispairingof TCR chains. For example, mispairing of an exogenous TCR a chain withan endogenous TCR β chain, or vice versa, can reduce the efficacy of theT cell therapy, and/or may result in the generation of autoimmuneresponses in patients. For example, the mixed exogenous/endogenous TCRpairings may trigger graft-versus-host disease (GVHD) or otherself-reactive responses. Thus, in some embodiments, the presentdisclosure encompasses host cells, compositions and methods forenhancing or ensuring the preferential pairing of exogenous TCR chainsand/or preventing pairing of exogenous chains with endogenous chains.Strategies for reducing mispairing are known in the art and includestrategies wherein the endogenous TCR is not disrupted and strategieswherein the presence or expression or activity of the endogenous TCR isdisrupted.

For example, TCRs in some embodiments, the preferential pairing ofexogenous TCR chains is achieved by adding one or more disulfide bondsto the constant regions of the exogenous TCR chains via introduction ofone or more cysteine residues. Exemplary cysteine substitutions to formdisulfide bonds are provided, for example, in U.S. Patent ApplicationPublication Nos. 2010-0047220 (U.S. Pat. No. 8,361,794) and2014-0112925, and PCT Publication No. WO 2016/70814, each of which isincorporated herein by reference in its entirety. Preferably, thecysteine residue substitution is selected from: Tyr10 to Cys, Ser15 toCys, Thr45 to Cys, Thr48 to Cys, R53 to Cys, and P89 to Cys of the alphachain; and/or Ser17 to Cys, Ala19 to Cys, Glu20 to Cys, Ser54 to Cys,Ser57 to Cys, Asp59 to Cys, Ser77 to Cys, and Ser78 to Cys of the betachain. In some embodiments, the cysteine residue substitution is at oneor more of the following pairs of locations: Thr48 of the alpha chainand Ser57 of the beta chain; Thr45 of the alpha chain and Ser77 of thebeta chain; Tyr10 of the alpha chain and Ser17 of the beta chain; Thr45of the alpha chain and Asp59 of the beta chain; or Ser15 of the alphachain and Glu15 of the beta chain.

In some embodiments, the preferential pairing of exogenous TCR chains isachieved by introducing non-human amino acid residues into the constantregion or by using a non-human amino acid constant region. Thus, in someembodiments, the present disclosure provides TCRs comprising humanvariable regions and non-human constant regions. Non-human constantregions may be constant regions from any mammal such as mice, rats, ornon-human primates. Non-human primates include, for example, gorillas,monkeys (e.g., rhesus monkeys, macaque monkeys), apes, chimpanzees,baboons, and orangutans. In some embodiments, the present disclosureprovides TCRs wherein murine amino acid residues have been introducedinto the constant region, such that the TCRs are fully or partiallymurinized.

In some embodiments, the preferential pairing of exogenous TCR chains isachieved by introducing mutations in the constant regions of the alphaand/or beta chains that provide steric associations that elicitpreferential pairing. For example, the TCR chains may have knob-and-holemutations. Exemplary knob-and-hole mutations are known in the art andinclude, for example, mutations and mutation strategies disclosed in USPatent Application Publication No. 2006-0166314, incorporated herein byreference in its entirety. In some embodiments, the knob-and-holemutation is generated by making one or more amino acid mutation thatresults in a sterically projecting group on one chain and a stericallyrecessed group on the other chain. Thus, in some embodiments, the stericinteractions of the two chains with the mutations result in preferentialpairing of the exogenous TCR alpha and beta chains.

In some embodiments, the preferential pairing of exogenous TCR chains isachieved by use of a physical linkage between the exogenous alpha andbeta chains. For example, the exogenously expressed chains can be linkedvia one or more linkers, such as a flexible peptide linker or pairedlinkers. In some embodiments, the exogenous TCR chains are paired viadimerization peptides fused to the exogenous TCR alpha and/or betachains.

In some embodiments, the present disclosure provides single chain TCRs,wherein the TCR alpha and beta chains of the single chain TCR arecomprised in a single polypeptide chain. Use of single chain TCRs alsoprevents mispairing of endogenous with exogenous TCR chains. In someembodiments, the single chain TCR comprises the variable alpha and betachains linked or conjugated to one another to form a single chain. Insome embodiments, the single chain TCR comprises an alpha chain variableregion fused to the N terminus of an alpha chain extracellular constantdomain sequence, and a beta chain variable region fused to the Nterminus of a beta chain extracellular constant and transmembranesequence, and a linker linking the alpha and beta chains. In otherembodiments, the single chain TCR comprises a beta chain variable regionfused to the N terminus of the beta chain extracellular constant domainsequence, and an alpha chain variable region fused to the N-terminus ofa beta chain extracellular constant and transmembrane sequence, and alinker linking the alpha and beta chains.

VI. Uses of TCRs

The TCRs described herein and/or TCRs identified by the methodsdescribed herein have a variety of uses including, but not limited to,as binding compounds for immunotherapy, as components of transfected Tcells for adoptive immunotherapy, as antigen sources in vaccines, and asindicators of immune status. For example, an individual complete TCRmolecule (e.g., comprising matched alpha and beta chains specific forthe same antigen) or a population of complete TCR molecules (e.g., twoor more complete TCR molecules that are specific for the same antigen orfor different antigens) can be provided in a soluble format may be usedas high affinity binding compounds linked to T cell capturing agents forunique anti-cancer therapeutics, e.g. as taught by Jakobsen et al, U.S.Pat. Nos. 7,329,731 and 7,666,604; which are incorporated herein byreference. In some embodiments, the soluble TCR comprises an alpha and abeta chain, wherein the transmembrane sequence in one or both chains hasbeen deleted. In some embodiments, the soluble TCR is a single chainsoluble TCR. A soluble single chain TCR, in some embodiments, comprisesthe variable alpha and variable beta chains fused or covalently linkedtogether through a linker peptide.

In some embodiments, the present disclosure provides a therapeuticvaccine for the treatment of cancer comprising a TCR provided herein. Insome embodiments, the vaccine comprise a nucleic acid encoding an alphachain TCR and/or a beta chain TCR provided herein. In some embodiments,the vaccines described herein are administered with an adjuvant. Commonadjuvants known in the art and suitable for use according to the presentdisclosure include, but are not limited to, alum (e.g., aluminumhydroxide, aluminum phosphate), emulsion-based formulations includingmineral oil, oligonucleotides (e.g., CpG DNA), bacterial cell wallcomponents (e.g., LPS), squalene, Freund's complete adjuvant, andFreund's incomplete adjuvant.

In some embodiments, nucleic acid sequences encoding matched TCR alphaand beta chains may be used to construct vectors which may, in turn, beintroduced to autologous T cells for use in adoptive immunotherapy of apatient. In some embodiments, samples from which TCRs are analyzed maybe taken before and after a patient has been immunized with a cancerantigen, so that elevated TCR chains specific for the cancer antigen arereadily matched and selected (e.g., by the methods described herein).References disclosing such applications include Turcotte et al, Adv.Surg., 45: 341-360 (2011); Morgan et al, Science, 314: 126-129 (2006);Walchli et al, PlosOne, 6: e27930 (2011); Robins et al, U.S. PatentApplication Publication No. 2010/0034834; and the like.

A population of matched or reconstituted TCRs from a sample comprises aunique profile of an individual's immune system, which contains muchmore information than profiles of single-sequence clonotypes. That is, apopulation of matched TCR chains or matched heavy and light chainimmunoglobulins comprises a clonotype profile where the clonotypes arepairs of nucleotide sequences that encode pairs of TCR chains expressedin the same T cell or pairs of heavy and light chain immunoglobulinsexpressed in the same B cell. In both cases, such pairs may be relateddirectly to T cell function, for example, by interaction with sets ofMHC tetramer-peptide complexes, e.g. Palmowski et al, Immunol. Rev.,188: 155-163 (2002); Hadrup et al, Nature Methods, 6: 520-526 (2009), orto B cell function, for example, by ELISAs, e.g. Reddy et al, NatureBiotechnology, 28(9): 965-969 (2010).

In one embodiment, clonotype profiles of matched immune receptor chainscomprise at least 100 clonotype pairs, wherein each clonotype of thepair comprises a sequence of from 30 to 300 nucleotides. In anotherembodiment, clonotype profiles of matched immune receptor chainscomprise at least 500 clonotype pairs, wherein each clonotype of thepair comprises a sequence of from 30 to 300 nucleotides. In anotherembodiment, clonotype profiles of matched immune receptor chainscomprise at least 1000 clonotype pairs, wherein each clonotype of thepair comprises a sequence of from 30 to 300 nucleotides. In stillanother embodiment, such clonotype profiles of matched immune receptorchains comprise pairs of TCR alpha and TCR beta clonotypes. In anotherembodiment, such clonotype profiles of matched immune receptor chainscomprise pairs of TCR gamma and TCR delta clonotypes.

The vectors may comprise a nucleic acid encoding an alpha chain TCRprovided herein, a beta chain TCR provided herein, or both an alpha anda beta chain TCR provided herein. In some embodiments, the vectors maycomprise nucleic acids encoding more than one alpha chain TCR providedherein, more than one beta chain TCR provided herein or more than onealpha chain TCR and more than one beta chain TCR provided herein. Insome embodiments, the vectors provided herein are capable of deliveringpolynucleotides to a host cell. A vector may be a viral vector, abacterial vector, a plasmid, a cosmid, a phage particle, an RNAmolecule, or a DNA molecule. In some embodiments, the vector is anexpression vector that has the ability to express the nucleic acidsprovided herein. In further embodiments, the expression vector maycomprise α nucleic acid that is operably linked to one or more controlsequences capable of effecting the expression of the nucleic acidmolecule in a host cell. Control sequences include transcriptionalpromoters, operator sequences, sequences encoding mRNA ribosome bindingsites, and other sequences involved in transcription and translation. Insome embodiments, the vector provided herein is a viral vector, such as,for example, a retrovirus or an adenovirus. In some embodiments, thevector is a lentiviral vector, parvoviral vector, or other retroviralvector. In some embodiments, the present disclosure provides a host cellcomprising the vector provided herein. In some embodiments, the hostcell comprises one or more vectors. For example, in some embodiments,the host cell comprises a vector comprising a nucleic acid encoding analpha chain TCR provided herein and a vector comprising a nucleic acidencoding a beta chain TCR provided herein.

For certain approaches to their use, one or more of the TCRs providedherein are exogenously expressed in a cell. Thus, in some embodiments,the present disclosure provides cells express one or more exogenous TCRs(e.g., an exogenous TCR specific for WT1 as provided herein). The cellmay be a stem cell, a hematopoietic progenitor cell, a peripheral bloodmononuclear cell (PBMC), and/or an immune cell. The immune cell may bean antigen presenting cell, dendritic cell, macrophage, Natural Killer(NK) cell, NK T cell, B cell, T cell, or any combination thereof. Insome embodiments, the cell is a T cell or an NK cell. The T cell may bea naïve T cell, an effector T cell, a central memory T cell, an effectormemory T cell, a CD4+ T cell, a CD8+ T cell, an alpha/beta T cell, agamma/delta T cell, a regulatory T cell, or any combination thereof. Insome embodiments, the T cell is a T cell that is capable of cytolyticactivity (CTL). T cells may be obtained from many sources includingPBMC, bone marrow, thymus, tissue biopsy, tumor, lymph node tissue, gutassociated lymphoid tissue, mucosa associated lymphoid tissue, spleentissue, or any other lymphoid tissue. Sources of T cells, or other cellswith cytolytic activity, may be autologous or allogenic, and may be froma single individual or from a population of individuals.

In some embodiments, the T cell is isolated from a subject. In someembodiments, the T cell is isolated from a sample collected from asubject. In some embodiments, the present disclosure provides methodsfor isolating T cells from a sample or subject and engineering the Tcells to exogenously express one or more TCR provided herein. In oneaspect, the isolated T cell is a CD4+ T cell or a CD8+ T cell. In aparticular embodiment, the T cell is a CD8+ T cell that nativelyexpresses at least one endogenous TCR. In some embodiments, the CD8+ Tcells have been engineered to decrease or eliminate endogenous TCRexpression.

In some embodiments, the T cell is a PD-1 knockout T cell. Morespecifically, in some embodiments, the T cell has been engineered tolack expression of programmed death ligand 1 (PD-1), which is aninhibitory signaling protein expressed on T cells. Knockdown of PD-1expression in the T cell may be achieved by any method for genesilencing or gene editing disclosed herein or any method known in theart, such as gene deletion, gene disruption, genome editing (via, forexample, ZFN, TALEN, or CRISPR/Cas nuclease systems), miRNA, siRNA,shRNA, or antisense approaches.

In some embodiments, the present disclosure encompasses recombinant hostcells, compositions and methods wherein endogenous TCR chain expressionis reduced or eliminated. For example, expression of the endogenous TCRsmay be inhibited or blocked by gene deletion, gene disruption, genomeediting, miRNA, siRNA, shRNA, or antisense approaches. Gene modificationmay be achieved using an engineered nuclease, such as a zinc fingernuclease (ZFN), TALE-nuclease (TALEN) system, or CRISPR/Cas nuclease.For example, zinc fingered proteins or TALEs may be fused to nucleasedomains to generate ZFNs and TALENs, which recognize their intendednucleic acid target through their engineered binding domains and causethe DNA to be cut near the ZFP or TALE via the nuclease activity. Inparticular embodiments, ZFN are used to selectively disrupt endogenousTCR alpha and beta chain expression. Methods for selectively disruptingendogenous TCR alpha and beta chain expression using ZFN technologiesare known in the art, for example, see U.S. Publication No.2011-0158957, which is incorporated herein by reference in its entirety.

In some embodiments, the cell comprising an exogenous TCR does notnatively or endogenously express any TCR. In other embodiments, the cellcomprising an exogenous TCR expresses a native or endogenous TCR only atlow levels, for example, levels that would not be sufficient to inducean immune response. In still other embodiments, the cell comprising anexogenous TCR expresses a native or endogenous TCR at levels similar tothose present in a wild type, non-engineered T cell.

In some embodiments, the TCRs and/or host cells and/or engineered Tcells provided herein are further engineered to express one or moreimmune modulating molecule. The immune modulating molecule may be animmune activating molecule. Thus, in some embodiments, the presentdisclosure provides compositions and methods comprising WT1-specificTCRs and/or cells exogenously expressing the WT1-specific TCRs, whereinthe TCRs and/or cells express an immune activating molecule to enhancethe immune response. The immune activating molecule may be a cytokine, aligand or receptor, a chemokine, a chemokine receptor, a costimulatorymolecule, or an antigen binding domain. For example, the molecule may beIL-12, TNFα, IFNγ, IL2, IL-7, IL-15, IL-2, IL-18, CD40L, or other immuneactivating cytokines known in the art. In some embodiments, the immuneactivating molecule may be selected from CD40, CD28, OX40, 4-1BBL, andligands thereof. In some embodiments, the immune activating molecule isCD40L, or 4-1BBL.

Methods for exogenously expressing immune activating molecules are knownin the art, for example, in U.S. Patent Publication No. 2016-0045551 andPCT Publication No. WO 2016/069283, which are incorporated herein byreference in their entireties. For example, cells known as chimericantigen receptor T (“CAR-T”) cells are T cells that exogenously expressan antigen binding protein (e.g., an scFv); “armored CAR-T cells” areCAR-T cells that further express an immune activating molecule. In someembodiments, the TCRs provided herein are “armored TCRs,” meaning thatthe TCRs are expressed in a cell along with an immune activatingmolecule. In some embodiments, the expression of the immune activatingmolecule may be exogenously expressed in the cell by way of introductionto the cell of an additional nucleic acid, other than the nucleic acidor nucleic acids encoding the TCR, for example, by way of a vector thatis separate and distinct from a vector comprising the TCR nucleic acidsequences. In other embodiments, the immune activating molecule may beexpressed on the same vector as the alpha and/or beta chain TCR.

In some embodiments, the present disclosure provides a cell comprisingeach of the TCR alpha and beta chain pairs provided herein. In someembodiments, the present disclosure provides a cell comprising more thanone exogenous TCR pair, wherein each exogenous TCR pair comprises adifferent mutation or strategy for ensuring or enhancing preferentialTCR pairing. For example, in some embodiments, the present disclosureprovides a cell comprising an exogenous TCR alpha and beta chain pairhaving complementary cysteine residue substitutions in the constantregion to provide a disulfide bond; an exogenous TCR alpha and betachain pair having all or part of the constant regions of TCR alpha andbeta chains that are non-human constant regions (e.g., murine ornon-human primate constant regions); an exogenous TCR alpha and betachain pair having complementary knob and hole mutations; an exogenousTCR alpha and beta chain pair wherein the alpha and beta chains arephysically linked together; an exogenous single chain TCR; and/or anycombination thereof. In some embodiments, two sets of paired exogenousTCR alpha and beta chains have different complimentary cysteine residuesubstitutions in the constant region. In some embodiments, theexpression of the endogenous TCR chains has been reduced or eliminatedby the genome editing, gene deletion, gene disruption, miRNA, siRNA,shRNA, or antisense approaches described herein, or by other means ofknocking down endogenous TCR expression known in the art.

In one aspect, the present disclosure provides microparticles ornanoparticles that comprise TCRs on the microparticle or nanoparticlesurface. In some embodiments, the TCR is present on the surface of themicroparticle or nanoparticle via linkage, such as by covalent linkage.In one embodiment, the nanoparticle is a non-biodegradable nanoparticle.In one embodiment, the nanoparticle is a polystyrene particle. In oneembodiment, the nanoparticle is a biodegradable particle. In oneembodiment, the nanoparticle is a PLGA nanoparticle or a citric acidnanoparticle. In one embodiment, the nanoparticle is a mesoporous silicananoparticle-supported liposome, where the nanoparticle is encapsulatedinside a liposome, with the TCR anchored to the liposome surface viachemical linking to a modified phospholipid. In some embodiments, themesoporous silica nanoparticle contains a cell toxic agent.

The present disclosure also provides for bispecific or multi-specificmolecules comprising a TCR provided herein. In further embodiments, theTCR is coupled to an antibody or fragment thereof. In furtherembodiments, the TCR is coupled to more than one antibody or fragmentthereof. The antibody or fragment thereof may be, for example, apolyclonal antibody, a monoclonal antibody, a Fab, scFv, or alternativeantigen binding scaffold. The antibody or fragment thereof may bespecific for a tumor antigen and/or a T cell molecule such as CD3. Insome embodiments, the bispecific or multi-specific molecules comprise αsoluble TCR provided herein linked to one or more antibody or fragmentthereof, wherein the TCR targets the antibody or fragment thereof toimmune cells to deliver a signal thereto. In some embodiments, thesignal is an immune activating signal. For example, in some embodiments,a TCR provided herein is linked to a CD3 specific Fab or scFv. TheTCR-Fab or TCR-scFv, in some embodiments, directs the lysis of tumorcells, thereby treating a cancer. In some embodiments, the TCR may becovalently linked through a peptide linker to the one or more antibodyor fragment thereof. In some embodiments, the TCR, bispecific molecule,or multi-specific molecule provided herein further comprises a drug ortherapeutic agent, and the molecule targets the drug or therapeuticagent to the cancer. For example, the TCRs provided herein may be usedto deliver a toxin or chemotherapeutic agent to the cancer.

In some embodiments, the present disclosure provides for pharmaceuticalcompositions comprising one or more recombinant TCRs described herein.In some embodiments, the pharmaceutical compositions further comprise αpharmaceutically acceptable carrier, diluent or excipient. As usedherein “pharmaceutically acceptable carrier, diluent or excipient”includes without limitation any adjuvant, carrier, excipient, glidant,sweetening agent, diluent, preservative, dye/colorant, flavor enhancer,surfactant, wetting agent, dispersing agent, suspending agent,stabilizer, isotonic agent, solvent, surfactant, and/or emulsifier whichhas been approved by the United States Food and Drug Administration asbeing acceptable for use in humans and/or domestic animals. Exemplarypharmaceutically acceptable carriers include, but are not limited to, tosugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate;tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal andvegetable fats, paraffins, silicones, bentonites, silicic acid, zincoxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil and soybean oil; glycols, such as propyleneglycol; polyols, such as glycerin, sorbitol, mannitol and polyethyleneglycol; esters, such as ethyl oleate and ethyl laurate; agar; bufferingagents, such as magnesium hydroxide and aluminum hydroxide; alginicacid; pyrogen-free water; isotonic saline; Ringer's solution; ethylalcohol; phosphate buffer solutions; and any other compatible substancesemployed in pharmaceutical formulations. Except insofar as anyconventional media and/or agent is incompatible with the agents of thepresent disclosure, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like. Further guidance regardingformulations that are suitable for various types of administration canbe found in Remington's Pharmaceutical Sciences, Mace PublishingCompany, Philadelphia, Pa., 17th ed. (1985). For a brief review ofmethods for drug delivery, see, Langer, Science 249:1527-1533 (1990).

VII. Kits and Vectors

Kits for detecting and/or treatment of WT1-expressing cancer are alsouseful and so are provided. In some embodiments, the kit comprises atleast one vector comprising at least one of the nucleic acids providedherein. In some embodiments, the kit comprises a nucleic acid comprisingat least one of SEQ ID NOs: 49-96, or a vector comprising a nucleic acidsequence according to at least one of SEQ ID NOs: 49-96. In someembodiments, the kit comprises a combination of one or more of SEQ IDNOs: 49-96. For example, in some embodiments, the kits provided by thepresent disclosure comprise at least 2, at least 3, at least 4, at least5, at least 6, at least 12, at least 18, at least 24, at least 30, or atleast 36 nucleic acid sequences selected from SEQ ID NOs: 49-96.

For example, in some embodiments, the kit comprises at least one nucleicacid sequence selected from SEQ ID NOs: 49-54, or a vector comprising atleast one nucleic acid sequence selected from SEQ ID NOs: 49-54. In someembodiments, the kit comprises at least 2, at least 3, at least 4, atleast 5, or 6 nucleic acid sequences (or a vector comprising the nucleicacid sequences) selected from SEQ ID NOs: 49-54. In some embodiments,the kit comprises at least one nucleic acid sequence selected from SEQID NOs: 57-62, or a vector comprising at least one nucleic acid sequenceselected from SEQ ID NOs: 57-62. In some embodiments, the kit comprisesat least 2, at least 3, at least 4, at least 5, or 6 nucleic acidsequences (or a vector comprising the nucleic acid sequences) selectedfrom SEQ ID NOs: 57-62. In some embodiments, the kit comprises at leastone nucleic acid sequence selected from SEQ ID NOs: 65-70, or a vectorcomprising at least one nucleic acid sequence selected from SEQ ID NOs:65-70. In some embodiments, the kit comprises at least 2, at least 3, atleast 4, at least 5, or 6 nucleic acid sequences (or a vector comprisingthe nucleic acid sequences) selected from SEQ ID NOs: 65-70.

In some embodiments, the kit comprises at least one nucleic acidsequence selected from SEQ ID NOs: 73-78, or a vector comprising atleast one nucleic acid sequence selected from SEQ ID NOs: 73-78. In someembodiments, the kit comprises at least 2, at least 3, at least 4, atleast 5, or 6 nucleic acid sequences (or a vector comprising the nucleicacid sequences) selected from SEQ ID NOs: 73-78. In some embodiments,the kit comprises at least one nucleic acid sequence selected from SEQID NOs: 81-86, or a vector comprising at least one nucleic acid sequenceselected from SEQ ID NOs: 81-86. In some embodiments, the kit comprisesat least 2, at least 3, at least 4, at least 5, or 6 nucleic acidsequences (or a vector comprising the nucleic acid sequences) selectedfrom SEQ ID NOs: 81-86. In some embodiments, the kit comprises at leastone nucleic acid sequence selected from SEQ ID NOs: 89-94, or a vectorcomprising at least one nucleic acid sequence selected from SEQ ID NOs:89-94. In some embodiments, the kit comprises at least 2, at least 3, atleast 4, at least 5, or 6 nucleic acid sequences (or a vector comprisingthe nucleic acid sequences) selected from SEQ ID NOs: 89-94.

In some embodiments, the kit comprises a first vector comprising a firstnucleic acid sequence encoding a TCR alpha chain and a second vectorcomprising a second nucleic acid sequence encoding a TCR beta chain,wherein the first nucleic acid sequence comprises at least 95%, 96%,97%, 98%, or 99% sequence identity to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 55, 63, 71, 79, 87, and 95, andthe second nucleic acid sequence comprises at least 95%, 96%, 97%, 98%,or 99% sequence identity to a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 56, 64, 72, 80, 88, and 96.

In some embodiments, the first nucleic acid sequence comprises at least95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 55 and thesecond nucleic acid sequence comprises at least 95%, 96%, 97%, 98%, or99% sequence identity to SEQ ID NO: 56. In some embodiments, the firstnucleic acid sequence comprises or consists of SEQ ID NO: 55 and thesecond nucleic acid sequence comprises or consists of SEQ ID NO: 56. Insome embodiments, the first nucleic acid sequence comprises at least95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 63 and thesecond nucleic acid sequence comprises at least 95%, 96%, 97%, 98%, or99% sequence identity to SEQ ID NO: 64. In some embodiments, the firstnucleic acid sequence comprises or consists of SEQ ID NO: 63 and thesecond nucleic acid sequence comprises or consists of SEQ ID NO: 64. Insome embodiments, the first nucleic acid sequence comprises at least95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 71 and thesecond nucleic acid sequence comprises at least 95%, 96%, 97%, 98%, or99% sequence identity to SEQ ID NO: 72. In some embodiments, the firstnucleic acid sequence comprises or consists of SEQ ID NO: 71 and thesecond nucleic acid sequence comprises or consists of SEQ ID NO: 72.

In some embodiments, the first nucleic acid sequence comprises at least95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 78 and thesecond nucleic acid sequence comprises at least 95%, 96%, 97%, 98%, or99% sequence identity to SEQ ID NO: 79. In some embodiments, the firstnucleic acid sequence comprises or consists of SEQ ID NO: 78 and thesecond nucleic acid sequence comprises or consists of SEQ ID NO: 79. Insome embodiments, the first nucleic acid sequence comprises at least95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 87 and thesecond nucleic acid sequence comprises at least 95%, 96%, 97%, 98%, or99% sequence identity to SEQ ID NO: 88. In some embodiments, the firstnucleic acid sequence comprises or consists of SEQ ID NO: 87 and thesecond nucleic acid sequence comprises or consists of SEQ ID NO: 88. Insome embodiments, the first nucleic acid sequence comprises at least95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 95 and thesecond nucleic acid sequence comprises at least 95%, 96%, 97%, 98%, or99% sequence identity to SEQ ID NO: 96. In some embodiments, the firstnucleic acid sequence comprises or consists of SEQ ID NO: 95 and thesecond nucleic acid sequence comprises or consists of SEQ ID NO: 96.

In some embodiments, the kit further comprises at least one cell. Insome embodiments, the kit comprises a cell expressing one or moreexogenous TCR alpha and/or beta chains according to any one of SEQ IDNOs: 7, 8, 15, 16, 23, 24, 31, 32, 39, 40, 47, or 48. In someembodiments, the kit comprises two different cells, wherein each cellexpresses a different TCR alpha and beta chain pair. For example, insome embodiments, the kit comprises a cell expressing an exogenous TCRalpha chain and an exogenous TCR beta chain according to SEQ ID NOs: 7and 8, respectively; another cell expressing an exogenous TCR alphachain and an exogenous TCR beta chain according to SEQ ID NOs: 15 and16, respectively; another cell expressing an exogenous TCR alpha chainand an exogenous TCR beta chain according to SEQ ID NOs: 23 and 24,respectively; another cell expressing an exogenous TCR alpha chain andan exogenous TCR beta chain according to SEQ ID NOs: 31 and 32,respectively; another cell expressing an exogenous TCR alpha chain andan exogenous TCR beta chain according to SEQ ID NOs: 39 and 40,respectively; and/or another cell expressing an exogenous TCR alphachain and an exogenous TCR beta chain according to SEQ ID NOs: 47 and48, respectively.

VIII. Therapy

The present disclosure provides methods for treating WT1-expressingcancer, wherein the methods comprise administering to a subject a cellcomprising one or more of the TCRs provided herein. In some embodiments,the method comprises obtaining cells (e.g., immune cells as providedherein) from the subject to be treated or from a cell donor source;introducing one or more nucleic acid encoding one or more of the TCRsprovided herein into the cells such that the cells exogenously expressthe TCRs provided herein; optionally further expanding the cells; andtransferring the cells to the subject. Thus, in some embodiments, themethods provided herein are autologous cell transfer methods, and inother embodiments the methods provided herein are allogenic celltransfer methods. In some embodiments, the cells transferred to thesubject comprise more than one TCR provided herein. For example, in someembodiments, the cells transferred to the subject are present in apopulation of cells wherein each cell comprises 1, 2, 3, 4, 5, or 6different exogenous TCRs. In other embodiments, the cells transferred tothe subject are present in a population of cells made up of 1, 2, 3, 4,5, or 6 subpopulations of cells, wherein each subpopulation of cellscomprises 1, 2, 3, 4, 5, or 6 different exogenous TCRs.

Cells comprising TCRs reactive against the WT1 oncoprotein epitope aresuitable for use in adoptive transfer methods to provide treatment to asubject in need of treatment for WT1-expressing cancer. The approach tosuch cell therapy generally comprises adoptively transferring to asubject in need thereof isolated cells expressing one or more of theTCRs provided herein under conditions permissive for expression of theTCR in the subject, as will be known to those of skill in the art. Thus,the present disclosure provides methods for treating WT1-expressingcancer, comprising adoptively transferring to a subject in need thereofisolated cells recombinantly expressing one or more of the TCRs providedherein.

In that respect, the present disclosure provides methods for adoptivetransfer of cells comprising the TCRs provided herein. Cells may beisolated from a subject using any method known in the art. For example,cells may be isolated using an isolation kit, Ficoll-Paque densitygradient centrifugation, flow cytometer cell sorting, and the like. Insome embodiments, isolated cells may be autologous (i.e., derived fromthe subject that will receive the resultant transduced or transformedcells). For example, the isolated cells may be obtained from PBMCsand/or hematopoietic stem cells of the subject. In other embodiments,isolated cells may be allogenic. In some embodiments, the isolated cellmay be an immune cell. The immune cell may be an antigen presentingcell, dendritic cell, macrophage, Natural Killer (NK) cell, NK T cell, Bcell, T cell, or any combination thereof. In certain embodiments, thecell is a T cell or an NK cell. The T cell may be a naïve T cell, aneffector T cell, a central memory T cell, an effector memory T cell, aCD4+ T cell, a CD8+ T cell, an alpha/beta T cell, a gamma/delta T cell,a regulatory T cell, or any combination thereof. In some embodiments,the isolated cell may be a T cell, such as a CD4+ T cell or a CD8+ Tcell. In particular embodiments, the isolated T cell is a CD8+ T cell.CD8+ T cells are also known as cytolytic T cells (CTLs). In someembodiments, the isolated T cells are expanded in vitro after separationfrom the subject. During expansion, the isolated T cells may beincubated with accessory cells (e.g., PBMC, dendritic cells, B cells, ormonocytes) to support expansion of the T cells in vitro prior totransfer to a subject. In some embodiments, unexpanded isolated T cellsare transferred, and such T cells may expand and become activated invivo.

For monitoring of therapy, the TCRs provided herein are associated witha therapeutic agent or detectable moiety. A detectable moiety may becovalently linked to one or both of the alpha and/or beta chains of theTCR. The detectable moiety may be a label suitable for diagnosticpurposes. For example, a detectable moiety may be an enzyme, a dye, anMRI-detectable reagent, a radionucleotide, or any suitable moiety knownto those skilled in the art. Such labelled TCRs are useful in a methodfor detecting a peptide-MHC complex (e.g., SEQ ID NO: 97-HLA-A*0201),for example for use in detection of antigen presenting cells carryingthe complex for which the high affinity TCRs provided herein arespecific.

In some embodiments, the present disclosure provides methods fortreating WT1-expressing cancer, wherein the methods compriseadministering to a subject a cell comprising one or more of the TCRsprovided herein. In some embodiments, the method comprises obtainingcells (e.g., immune cells as provided herein) from the subject to betreated or from a cell donor source; introducing one or more nucleicacid encoding one or more of the TCRs provided herein into the cellssuch that the cells exogenously express the TCRs provided herein;optionally further expanding the cells; and transferring the cells tothe subject. Thus, in some embodiments, the methods provided herein areautologous cell transfer methods, and in other embodiments the methodsprovided herein are allogenic cell transfer methods. In someembodiments, the cells transferred to the subject comprise more than oneTCR provided herein. For example, in some embodiments, the cellstransferred to the subject are present in a population of cells whereineach cell comprises 1, 2, 3, 4, 5, or 6 different exogenous TCRs. Inother embodiments, the cells transferred to the subject are present in apopulation of cells made up of 1, 2, 3, 4, 5, or 6 subpopulations ofcells, wherein each subpopulation of cells comprises 1, 2, 3, 4, 5, or 6different exogenous TCRs.

In some embodiments, the treatment methods provided herein compriseadministering to a subject about 10⁵, about 10⁶, about 10⁷, about 10⁸,about 10⁹, or about 10¹⁰ cells exogenously expressing the TCRs providedherein. In some embodiments, the treatment methods provided hereincomprise administering to a subject about 10⁵ to about 10¹⁰ cellsexogenously expressing the TCRs provided herein, or about 10⁶ to about10⁹ cells exogenously expressing the TCRs provided herein, or about 10⁷to about 10⁸ cells exogenously expressing the TCRs provided herein. Insome embodiments, the methods provided herein comprise administering tothe subject about 10⁵ cells/kg body weight, about 10⁶ cells/kg bodyweight, about 10⁷ cells/kg body weight, about 10⁸ cells/kg body weight,about 10⁹ cells/kg body weight, or about 10¹⁰ cells/kg body weight. Insome embodiments, the methods provided herein comprise administering tothe subject about 10⁵ cells/kg body weight to about 10¹⁰ cells/kg bodyweight, or about 10⁶ cells/kg body weight to 10⁹ cells/kg body weight,or about 10⁷ cells/kg body weight to about 10⁸ cells/kg body weight. Insome embodiments, the methods comprise administering the cells to thesubject in a single dose or in multiple doses. Administration of thecells to the subject may be performed parenterally, for example byintravenous infusion.

In some embodiments, cells expressing one or more of the TCRs providedherein are administered to a subject prior to, concurrently with, orsubsequent to an additional therapeutic treatment for cancer. Forexample, in some embodiments, cells expressing one or more of the TCRsprovided herein are administered to a subject prior to, concurrentlywith, or subsequent to a chemotherapeutic agent and/or a biologicalcancer therapy. Chemotherapeutic agents may include, but are not limitedto, cyclophosphamide, doxorubicin, hydroxydaunomycin, vincristine,taxanes (e.g., paclitaxel), and others known in the art. Anti-viralagents may include, but are not limited to, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, protease inhibitors, viral fusion inhibitors, andinterferons such as IFNα.

In particular, for use in animal models, the sequence of the TCR a and Rchains may be selected based on species. In some embodiments, transgenicanimals expressing human MHC molecules may also be useful in evaluatingspecific aspects of the present invention.

While the present invention has been described with reference to severalparticular example embodiments, those skilled in the art will recognizethat many changes may be made thereto without departing from the spiritand scope of the present invention. The present invention is applicableto a variety of sensor implementations and other subject matter, inaddition to those discussed above.

EXAMPLES Example 1

In this example steps common to some embodiments, such as the embodimentof FIG. 12C, are described for particular applications, including butnot limited to, exposing a tissue sample comprising T cells to antigen,activating T cell in a tissue sample by antigen, obtaining recombinednucleic acids from T cells of a tissue sample, isolating (or recovering,or sorting, or separating) activated T cells, sequencing recombinednucleic acids, forming clonotypes, and determining clonotypes ofantigen-specific T cells.

Tissue Samples:

Characterized PBMCs were purchased from Cellular Technology Limited.Cells were thawed, washed and either lysed with RLT plus buffer (Qiagen)for nucleic acid purification or cultured overnight in the presence ofpeptides (see below) to identify antigen-specific T cells.

Antigen-Specific T Cell Assays, Flow Cytometry and Cell Sorting:

Antigen-specific cells were identified using a variety of assays:pentamer binding, cell surface marker upregulation (CD137, CD107)following short-term peptide incubation, and proliferation followingrelatively long-term peptide incubation. Pentamer-specific T cells wereidentified by incubating PBMCs with HCMV pp65₄₉₅-504 Pentamer(ProImmune) according to manufacturer's instructions. The procedures forobtaining viable antigen-specific T cells based on acquisition of thecell surface markers CD137/107 (for CD8 antigen-specific T cells)following brief in vitro incubation with peptides are well-known, e.g.Chattopadhyay et al, Nature Medicine, 11: 1113-1117 (2005); Meier et al,Cytometry A, 73: 1035-1042 (2008); Wolfl et al, Blood, 110: 201-210(2007); Wolfi et al, Cytometry A, 73: 1043-1049 (2008); and the like.

Briefly, complete media containing 15% Fetal Bovine Serum (FBS),non-essential amino acids, glutamine and antibiotics was used forpeptide incubations. Thawed PBMCs were washed and suspended at−400,000cells/well (96-well i-bottom plates) in complete media. Unconjugatedantibodies directed against CD28 and CD49d were then added to the wellscontaining the suspended cells. Peptides derived from CMV pp65 (HCMVA(pp65) (JPT Peptide Technologies) were added directly to thecell/antibody mixture, according to manufacturer's instructions. Asingle peptide derived from CMV pp65 (sequence NLVPMVATV (SEQ ID NO:98); herein referred to as “pp65₄₉₅”) was used at 2 μg/mL. pp65 “PepMix”and CEF+ peptide pools (JPT Peptide Technologies) were added directly tothe cell/antibody mixture, according to manufacturer's instructions.Following addition of peptides, cells were incubated at 37° C. for ˜ 8hours. Negative control incubations were prepared as outlined abovewithout addition of peptides.

At the end of the incubation, cells were harvested from the culture andstained with antibodies for analysis and sorting by flow cytometry. Foreach CD8 antigen-specific assay (CD137 and CD107), fluorescentlyconjugated antibodies to the following cell surface markers were usedfor flow cytometry: CD8, CD3 and either CD137 or CD107a and CD107b.Cells were then washed and suspended in PBS containing FBS (2%) and4′,6-diamidino-2-phenylindole (DAPI) for exclusion of non-viable cells.Carboxyfluorescein diacetate, succinimidyl ester (CFSE)-labeled PBMCswere incubated as outlined above for 6 days in the presence of peptideand antibodies directed against CD28 and CD49d. Antigen-specific CD8+ Tcells were identified and sorted based on CFSE dilution at day 6. Cellswere acquired and sorted using a FACSAria (BD Biosciences) instrument.Sorted antigen-specific (CD3⁺CD8⁺CMV pentamer⁺, CD3⁺CD8⁺CD137⁺,CD3⁺CD8⁺CD107a/b⁺, or CD8⁺CFSE^(low)) and non-antigen-specific(CD3⁺CD8⁺CD137, CD3⁺CD8⁺CD107a/b⁻) cells were pelleted and lysed in RLTPlus buffer for nucleic acid isolation. Analysis of flow cytometry datafiles was performed using FlowJo (Ashland, Oreg.).

RNA and cDNA Preparation:

RNA (and DNA) was isolated using AllPrep DNA/RNA mini and/or micro kits,according to manufacturer's instructions (Qiagen). RNA was reversetranscribed to cDNA using Vilo kits (Life Technologies).

TCR Beta Amplification and Sequencing.

cDNA was amplified using locus specific primer sets for TCR beta. Thisamplification reaction reproducibly amplified all possible RNAtranscripts found in the sample containing the rearranged TCR beta locusregardless of which variable (V) segment and which common constant (C)region allele each rearranged molecule possessed, while appending thenecessary sequences for cluster formation and sample indexing.

First stage primers were designed so as to allow for the amplificationof all known alleles of the germline sequences, as described above andin the following; Faham et al, Blood, 120: 5173-5180 (2012). At the 5′ends of the V segment primers, universal sequences complementary tosecond stage PCR primers were appended. Primers were optimized such thateach possible V and C segment was amplified at a similar rate so as tominimally skew the repertoire frequency distribution during theamplification process. Specificity of the primers was, in contrast, notoptimized as the primer sequences could be mapped and removed from theeventual sequence read. Thus, a given sequence may have been amplifiedby multiple primers.

In the second stage PCR, primers on the C side annealed to the C segmentwith a 5′ tail that contained the sequence primer and the P5 sequenceused for cluster formation in the Illumina Genome Analyzer sequencer.Primers on the V side annealed to the V segment with a 5′ tail thatcontained the sequence primer and the P7 sequence used for clusterformation. For each sample, one pair of primers is used in the secondstage. On the C side, it is always the same primer. On the V side, it isone of a set of primers which differs by a 6 base index. Specifically,the primers on the V side of the amplification constituted one of a setof primers, each of which had a 3′ region that annealed to the overhangsequence appended in the first reaction but which further contained oneof multiple 6 base pair indices that allowed for sample multiplexing onthe sequencer. Each of these primers further contained a 5′ tail withthe P7 sequence used for cluster formation in the Illumina GenomeAnalyzer sequencer.

First stage PCR was carried out using a high-fidelity polymerase(AccuPrime, Life Technologies) for 16 cycles. A second stage PCR wascarried out for 22 cycles on 1/100 of the amplification products fromthe first stage PCR. Each sample contained a unique identifying tag.Samples were pooled and purified using the QlAquick PCR purification kit(Qiagen). Cluster formation and sequencing in both directions wascarried out per the manufacturer protocol (Illumina, Inc., La Jolla,Calif.). Specifically, three sequencing reactions were performed. First115 bp were sequenced from the C side sufficient to sequence through thejunctional sequence from C to V. At this point, the synthesized strandwas denatured and washed off. A second sequencing primer was annealedthat allowed the sample index to be sequenced for 6 cycles to identifythe sample. At this point the reverse complement strand was generatedper the Illumina protocol. A final sequencing read of 95 bp was obtainedfrom the V-to-C direction providing ample sequence to map the V segmentaccurately. The sequencing data was then analyzed to determine theclonotype sequences, as described above.

Clonotype Determination:

A clonotype was defined when at least 2 identical sequence reads wereobtained. Briefly, after exclusion of low quality reads, sequence datawere then analyzed to determine the clonotype sequences includingmapping to germline V and J consensus sequences. First, the sample indexsequences were used to identify which of the sequences originate fromwhich of the pooled samples. Sequences whose index were not a perfectmatch to one of the indices used in a specific run were excluded. Nextthe forward read was used to map the J segment. Since all the sequencesstarted from the same position of the J segments, all the J segmentsstarted at a predefined sequencing position. The first 25 bp of the Jsegments were used to map the J segment. Any read with more than 5 highquality mismatches to the known J segments was excluded from furtheranalysis.

After J segment identification, V segments were mapped. The reverse readwas used for this purpose. First, the V primer was mapped and excluded.Thereafter, the next 70 bases of the reverse read were mapped to theknown V segments. Reads that did not map to J and V segments wereexcluded. The next step in mapping involved identifying the frame thatrelated the forward and reverse reads and this allowed a continuoussequence from J to V to be constructed. This was done using the last 15bases of the forward read which were reliably within the V segmentregardless of NDN length. While these bases could be of relatively lowersequence quality as they were at the terminal end of a long read, theycould be used to map within a single identified V segment in order toidentify the position at which the two reads could be joined. Finally,the known V and J sequences to which the reads map were used to identifythe point in the forward read at which the sequences at the junctionsdiverged from these mapped segments.

Following clonotype determination, relative frequencies of theclonotypes were analyzed in the unsorted, antigen-specific andnon-antigen-specific populations. Clonotype frequency comparisonsbetween two samples are shown in several figures. Clonotypes that arepresent in sample A but not in sample B (where frequencies in sample Aand B are being compared) are represented to have the frequency of aclonotype with a single read in sample B. Therefore the frequency of themissing clonotype in a sample depends on the sequencing depth of aparticular sample. In these cases where a clonotype is missing in asample, because the frequency of a single read is assigned to theseclonotypes, the observed frequency is overestimated. Thus, in the vastmajority of these cases, the real clonotype frequency is likely to beoverestimated. Clonotypes absent in both samples appear where the axesintersect. Clonotypes present in one sample but not the other howeverlie along either the x- or y-axis.

Clonotypes from the antigen-specific T cell analyses were selected basedon three criteria. First, selected clonotypes had a frequency in sortedantigen-specific populations that was increased by at least 10-fold overthe frequency in non-antigen-specific or unsorted cell populations (e.g.FIGS. 17A and 17B). Second, these clonotypes were also present at lowerfrequencies in sorted, non-antigen specific cells compared to unsortedcells if greater than 1/100,000 in order to avoid sub-sampling error(Poisson noise) associated with very low frequency clonotypes in sortedsamples. Third, because of the limited number of input antigen-specificcells after sorting (generally <30,000 cells), a greater than 20-cellequivalent threshold was applied based on the relatively low inputnumber of cells in these samples. This minimum threshold enabledexclusion of clonotypes that appeared enriched in sortedantigen-specific samples but were due only to the presence of one or afew cells in the sample. For example, consider a sorted population of10,000 pentamer⁺ cells out of a sample with a million T cells. If asingle cell with a frequency of 1 per million in the unsorted sample isincidentally sorted in the pentamer+ sample, its frequency in the sortedsample will be 1/10,000 and would appear to be 100 fold enriched in thepentamer⁺ sample compared to the unsorted sample. To ameliorate thisproblem, a clonotype was required to represent 20 cells in the sortedpentamer+ sample. Specifically, the log₁₀ frequency threshold requiredin the pentamer+ sample was calculated as log₁₀(1/(n/20)), where n isthe number of input sorted cells for that sample as determined by flowcytometry (For example, in FIG. 18A, 16, 281 is number of input sortedcells and the calculated threshold frequency is 10²⁻⁹). Those sequencesmeeting the three criteria outlined above were classified asantigen-specific T cell clonotypes.

Results:

The combination of sorting and sequencing was used to identifyantigen-specific clonotypes in an individual with a known positiveresponse to a cytomegalovirus (CMV) antigen. First, TCR beta sequencingwas paired with a multimer-based immune assay to validate this methodfor identification of antigen-specific CD8 TCR beta clonotypes. Apeptide derived from CMV pp65(495-404) (pp65₄₉₅ peptide) is anHLA-A*0201-restricted immunodominant epitope that induces responses inCMV-positive individuals. To directly identify T cells specific to thisantigen, a commercially available pentamer reagent containing pp65₄₉₅peptide bound to an MHC molecule was used. In principle, all of the Tcells carrying the sequences that bind the pentamer should be detectedirrespective of their functional potential. pp65₄₉₅-specific CD8 T cellswere identified by sequencing the TCR beta repertoire of cells that weresorted based on pentamer binding (pentamer⁺).

Frozen PBMCs were obtained from an individual with a characterizedresponse to pp65₄₉₅ by ELISPOT assay. Two populations were sorted fromthis individual: CD8 pentamer+ and pentamer T cells. Nucleic acidsencoding TCR beta clonotypes were sequenced in these two populationsalong with the unsorted PBMC sample, so that the relative frequencies ofthe clonotypes in each population could be determined. Three criteriawere used to identify pp65₄₉₅-specific TCR beta clonotypes: 1)Clonotypes that are enriched (i.e. have substantially higher frequency)in the pentamer+ population compared to the pentamer population; 2)Clonotypes that are enriched in the pentamer+ population compared to theunsorted sample; and 3) Clonotypes that are de-enriched (i.e. have lowerfrequency) in the pentamer population compared to the unsorted sample.

Eight clonotypes were identified that are substantially enriched (−1,000fold) in the pentamer+ compared to the pentamer population (FIG. 17A).The frequencies of these clonotypes were compared in the pentamer+ andthe unsorted populations (FIG. 17B). The highest of these clonotypes hada frequency of 0.81% in the unsorted sample, which is consistent withthe expected elevated response to pp65₄₉₅ in this individual. However,several of the other clonotypes were present at a level below 10⁻⁴. The8 clonotypes are enriched in the pentamer⁺ population by a factor of−100fold compared to their frequency in unsorted PBMC.

PBMCs from the same individual were used to assess whether immune assaysthat rely on indirect or functional changes in the T cells followingantigen stimulation are effective for identification of pp65₄₉₅-specificCD8 TCR beta clonotypes. PBMCs were stimulated with pp65₄₉₅ followed byflow cytometry 18 hours after the stimulation to capture cells based onexpression of the activation marker CD137. The TCR beta repertoire wasamplified and sequenced from sorted CD137⁺ and CD137⁻ cells. Thecriteria for identification of pp65₄₉₅-specific TCR beta clonotypes withthis assay was similar to that used in the pentamer assay. Specifically,pp65₄₉₅-specific TCR beta clonotypes were expected to be present at muchhigher frequencies in the CD137⁺ population compared to the CD137⁻population.

Nine clonotypes were identified that are substantially enriched (−1,000fold) in the CD137⁺ population compared to the CD137⁻ population (FIG.18A). The frequency of these clonotypes in the unsorted sample rangedfrom as high as 0.81% to as low as 0.004% (FIG. 18B). These clonotypeswere enriched in the CD137⁺ population compared to the unsorted PBMCsample by −100 fold. To ensure that these cells were activated due tostimulation with the peptide, a control experiment was performed with nopeptide. None of the 9 clonotypes that were enriched with the peptide inthe CD137⁺ population enriched following incubation without peptide inCD137⁺ cells (FIG. 18C).

Specific clonotypes identified by the pentamer and CD137 assays werecompared and found to substantially overlap. All 8 clonotypes that wereidentified with the pentamer assay were also identified by CD137 assay(FIG. 19A), although an additional clonotype was identified by the CD137assay that was not identified in the pentamer assay.

A third functional assay for identification of antigen-specificclonotypes was conducted by combining capture of proliferating cellsfollowing incubation with pp65₄₉₅ peptide and repertoire sequencing.Cells were labeled with CFSE and incubated with either pp65₄₉₅ or nopeptide for 6 days. Proliferating CD8 cells were then sorted based ondilution of CFSE. pp65₄₉₅-specific clonotypes were identified based ontheir relative frequency in the CFSE^(low) population compared to thatof fresh unsorted PBMCs.

Sixteen clonotypes were identified that were substantially increased inthe CFSE^(low) population, and the frequency of some of the identifiedclonotypes was below 10⁻⁵ (FIG. 20A). An identical proliferation assaywas used that lacked the peptide as a control. None of the 16 clonotypesidentified by the proliferation assay were enriched in the CFSE® Wpopulation when no peptide was used (FIG. 20B).

One advantage to using indirect immune monitoring assays compared topentamer reagents is the ability to assess responses to more than onepeptide antigen at the same time. A pool of 138 peptides spanning theentire pp65 protein (herein referred to as pp65pool) was used tostimulate PBMCs in the proliferation assay to identify pp65pool-specificT cells. Repertoire analysis of proliferating cells following pp65poolincubation enabled identification of 25 clonotypes. Of these 25clonotypes identified using the pp65pool, 12 of these were also deemedantigen-specific with the single pp65₄₉₅ peptide, demonstrating therepeatability of the approach.

Seven of eight clonotypes identified by the pentamer assay wereidentified in the pp65pool proliferation assay, demonstrating that theuse of peptide pool does not substantially decrease sensitivity. Inaddition the proliferation assay with the pp65pool enabledidentification of additional clonotypes that are presumably specific toother peptides within the pool. Most of the additional clonotypesidentified with the pp65pool were not enriched in the pentamer⁺population (FIG. 20D) consistent with them being not specific to thepp65₄₉₅ peptide.

Example 2: Generation of TCRs Against WT1

Bulk naïve T cells were isolated from normal healthy donor peripheralblood mononuclear cells (PBMC) and expanded with anti-CD3/CD28 with IL-2for 11 days. Cells were incubated with a WT1-derived peptide,(YMLDLQPETT; SEQ ID NO: 97, WT1-pep). Antigen-specific T cells weresorted based on expression of CD3, CD8, and CD107 and/or CD137. Nucleicacids were prepared from both positive (CD107⁺ and/or CD137⁺, i.e.,antigen-specific) and negative (CD107⁻ and/or CD137⁻) T cells. The TCRαand TCRβ repertoires were amplified and sequenced using next generationsequencing.

Methods for immune repertoire profiling are described in U.S. Pat. No.8,236,503, PCT International Publication Nos. WO 2010/151416 and WO2011/106738, U.S. Patent Application Publication Nos. 2014/0256567 and2012/0058902, which are each incorporated by reference in theirentireties. In some embodiments, methods and compositions forcontrolling amplification bias in a single multiplex PCR are used asdescribed in U.S. Pat. No. 9,150,905, which is incorporated by referencein its entirety.

Nucleotide sequences for the exemplary TCRα and TCRβ chains are providedbelow in Tables 1 and 2.

TABLE 1 CDR sequences of WT1 TCRs SEQ TCR ID Name Chain CDRAmino Acid Sequence NO: eJH30_5 TCRα CDR1 DSASNY 1 CDR2 IRSNVGE 2 CDR3CAAGGRDDKIIF 3 TCRβ CDR1 SNHLY 4 CDR2 FYNNEI 5 CDR3 CASSERLGTMAYN 6SPLHF eJH30_8 TCRα CDR1 SIFNT 9 CDR2 LYKAGEL 10 CDR3 CAGRGSQGNLIF 11TCRβ CDR1 LGHDT 12 CDR2 YNNKEL 13 CDR3 CASSHWQETQYF 14 eJH52_13 TCRαCDR1 TSENNYY 17 CDR2 QEAYKQQN 18 CDR3 CAFMGYYGGSQG 19 NLIF TCRβ CDR1SGHNS 20 CDR2 FNNNVP 21 CDR3 CASSSLQYEQYF 22 eJH64_9 TCRα CDR1 NSASQS 25CDR2 VYSSGN 26 CDR3 CVVKSLDNNNDMRF 27 TCRβ CDR1 KGHDR 28 CDR2 SFDVKD 29CDR3 CATSDWTGRNEQFF 30 eJH64_6 TCRα CDR1 VSGNPY 33 CDR2 YITGDNLV 34 CDR3CAVRDMRYGGAT 35 NKLIF TCRβ CDR1 LGHNA 36 CDR2 YSLEER 37 CDR3CASSQDGLAGAAS 38 FNNEQFF eJH30_7 TCRα CDR1 SSNFYA 41 CDR2 MTLNGDE 42CDR3 CAFMRATGANNLFF 43 TCRβ CDR1 SGHVS 44 CDR2 FNYEAQ 45 CDR3CASSFGGVSYEQYF 46

TABLE 2 Full TCRα and TCRβ chain sequences of WT1 TCRs SEQ Name RegionAmino Acid Sequence ID: eJH30_5 TCRαMTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSA 7SNYFPWYKQELGKGPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAAGGRDDKIIFGKGTRLHILPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS TCRβMDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNH 8LYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSERLGTMAYNSPLHFGNGTRLTVTEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF eJH30_8 TCRαMLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSSIFN 15TWLWYKQEPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGRGSQGNLIFGKGTKLSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS TCRβMGCRLLCCVVFCLLQAGPLDTAVSQTPKYLVTQMGNDKSIKCEQNLGH 16DTMYWYKQDSKKFLKIMFSYNNKELIINETVPNRFSPKSPDKAHLNLHINSLELGDSAVYFCASSHWQETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATL YAVLVSALVLMAMVKRKDSRGeJH52_13 TCRα MTRVSLLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTS 23ENNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFMGYYGGSQGNLIFGKGTKLSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS* TCRβMDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGH 24NSLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSSLQYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKAT LYAVLVSALVLMAMVKRKDSRGeJH64_9 TCRα MISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSN 31SASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVKSLDNNNDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS* TCRβMASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGH 32DRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSDWTGRNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKA TLYAVLVSALVLMAMVKRKDSRGTCRα MASAPISMLAMLFTLSGLRAQSVAQPEDQVNVAEGNPLTVKCTYSVSG 39NPYLFWYVQYPNRGLQFLLKYITGDNLVKGSYGFEAEFNKSQTSFHLKKPSALVSDSALYFCAVRDMRYGGATNKLIFGTGTLLAVQPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS* eJH64_6 TCRβMGCRLLCCAVLCLLGAGELVPMETGVTQTPRHLVMGMTNKKSLKCEQH 40LGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPSRFSPECPNSSHLFLHLHTLQPEDSALYLCASSQDGLAGAASFNNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILY EILLGKATLYAVLVSALVLMAMVKRKDSRG TCRαMEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFP 47SSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCAFMRATGANNLFFGTGTRLTVIPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS* eJH30_7 TCRβMGTSLLCWVVLGFLGTDHTGAGVSQSPRYKVTKRGQDVALRCDPISGH 48VSLYWYRQALGQGPEFLTYFNYEAQQDKSGLPNDRFSAERPEGSISTLTIQRTEQRDSAMYRCASSFGGVSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG *indicates presence of stop codon

Example 3. Target PMHC Off-Rate Kinetics

The WT1 peptide antigen used in this experiment is YMLDLQPETT (SEQ IDNO: 97, WT1-pep). This peptide binds to HLA A*02.01 by netMHC with anaffinity of about 176 nM.

Tetramer off rate assay: TCR-deficient Jurkat (JRT3) cells wereelectroporated with either the eJH30_WT1C_5 (FIG. 1A) or theeJH130_WT1C_8 (FIG. 1B) TCR gene constructs and then stained withWT1-pep/HLA-A2-PE tetramers. Initial tetramer fluorescence intensity wasassessed by FACS, after which anti-HLA-A2 blocking antibody (BB7.2) wasadded to prevent tetramer re-binding to cells, and fluorescenceintensity was re-assessed every 15 seconds for 3 minutes. Tetramerdissociation overtime was plotted as the natural logarithm of tetramerfluorescence intensity (minus background) over time. The slope of thisline provided the negative of the dissociation rate (−kd), and(ln(2))/kd) represents the half-life (t_(1/2)) of WT1/HLA-A2 tetramer'sinteraction with TCR on the cells.

The results of the study are provided in Table 3 and FIGS. 1A and 1B.

TABLE 3 Tetramer −kd, tetramer half-life, and tetramer initial MFI forWT1 TCRs Tetramer Tetramer TCR ID −kd t_(1/2) (s) initial MFIeJH30_WT1C_5 −0.003315 209 3216 eJH30_WT1C_8 −0.003729 176 4132

Example 4. Functional Assessment of Wt1 TCRs

A functional avidity assay was carried out to assess the TCRs' relativepotency. TCR-deficient Jurkat cells (JRT3) were electroporated withvectors encoding the indicated TCRs and allowed to rest overnight.TAP-I-deficient T2 cells were pulsed with the indicated concentrationsof WT1-pep for 30 minutes, washed, and then mixed with JRT3s expressingthe TCRs described herein for 4 hours. Cells were stained for CD3 (as ameasure of TCR expression) and CD69, then analyzed by FACS. CD69 MFI ofthe TCR⁺ JRT3 cells was plotted against peptide concentration to assessthe TCRs' relative potency. The results of these experiments are shownin FIG. 2 . The mean EC₅₀ (nM) for each peptide is provided in Table 4.

TABLE 4 EC₅₀ of functional avidity for WT1 TCRs TCR ID EC₅₀ (nM)eJH30_WT1C_5 51.85 eJH30_WT1C_8 10.88

A cytolysis assay was performed to assess the antigen-specific killingcapacity of the WT1 TCRs. CD8⁺ T cells were isolated from PBMCs fromhealthy donors, then expanded 7 days using anti-CD3/anti-CD28 and IL-2.Expanded T cells were electroporated with RNA encoding the indicatedTCRs and allowed to rest 4 hours. TAP-I-deficient T2 target cells (T)were pulsed either with 10 μg/mL WT1-pep or with the vital dyeCellTrace-647, then mixed in a 50:50 ratio. CD8⁺ T cells (effectorcells; E) were then added to the target cells at the indicated ratios ofCD8⁺ T cell effector cells to antigen⁺ T2 target cells, then incubated16 hours. Cells were then analyzed by FACS, and the remaining percentageof antigen⁺ T2 cells (relative to the control CellTrace⁺ T2 cells) wasplotted against E:T ratio, as a measure of antigen-specific killingcapacity.

An additional cytolysis assay was performed with peptide titration toassess the relative antigen-specific killing potency of each peptide.CD8⁺ T cells were isolated from PBMCs from healthy donors, then expanded7 days using anti-CD3/anti-CD28 and IL-2. Expanded T cells wereelectroporated with RNA encoding the indicated TCRs and allowed to rest4 hours. TAP-I-deficient T2 target cells were pulsed either with 10μg/mL of WT1-pep or with the vital dye CellTrace-647, then mixed in a50:50 ratio. CD8⁺ T cells (effector cells; E) were then added to thetarget cells (T) at a ratio of 2:1 E:T (2:1 CD8⁺ T cells:antigen+T2cells) and then incubated 16 hours (FIG. 3 ).

In a separate experiment, TAP-I-deficient T2 target cells were pulsedeither with the indicated concentrations of WT1-pep or with the vitaldye CellTrace-647, then mixed in a 50:50 ratio. CD8⁺ T cells (effectorcells; E) were then added to the target cells (T) at a ratio of 2:1 CD8⁺T cells:antigen⁺ T2 cells, then incubated 16 hours (FIG. 4 ). In eachexperiment, cells were analyzed by FACS, and the remaining percentage ofantigen⁺ T2 cells (relative to the control CellTrace⁺ T2 cells) wasplotted against peptide concentration, as a measure of antigen-specifickilling potency.

Example 5. Treatment of Leukemia with Cells Exogenously Expressing TCRsSpecific for WT1

A study is conducted to assess the efficacy of the TCRs provided hereinin the treatment of patients having leukemia. In some aspects of thestudy, T cells will be isolated from PMBCs of patients that have beendiagnosed with leukemia and are positive for HLA-A*0201, HLA-A*0203,and/or HLA-A*0206, for an autologous transfer of the cells back to thepatient after cell modification. In other aspects of the study, the Tcells will be donor T cells from a donor source that is positive forHLA-A*0201, HLA-A*0203, and/or HLA-A*0206, for an allogenic celltransfer to the patient after cell modification.

Vectors comprising the TCR alpha and beta chains provided herein will beintroduced into the T cells to generate T cells exogenously expressingthe WT1-specific TCRs provided herein. The autologous or allogenic cellswill be infused into the patients. Patients will receive about 10⁵, 10⁶,10⁷, 10⁸, or 10⁹ autologous or allogeneic cells per kg body weight.

Patients will be monitored for adverse events, survival and relapserates, clinical response, and durability of clinical response. Bloodtests, CT scans, skin biopsies, and bone marrow biopsies may beconducted to monitor disease progress, tumor size, and immune responsesin the patients.

The results of the study will show that the TCRs provided herein providea safe and effective means of inducing immune responses against WT1 andtreating cancer.

Example 6: Functional Assessment of WT1 TCRs

Cytolysis and cytokine expression assays were performed to assess theantigen-specific killing capacities of the following WT1 TCRs: eJH52_13,eJ64_9, eJH64_6, and eJH30_7. The amino acid sequences of each of theseTCRs are provided above in Tables 1 and 2.

Endogenous, presentation-deficient T2 cells were incubated withincreasing concentrations of WT1 peptide. The cells were then incubatedfor 16 hours with polyclonal T cells expressing either no exogenous TCR(FIG. 5 , polyclonal T cells), or expressing the indicated WT1-specificTCR. For the cytolysis study, following incubation, the remaining viablepeptide-loaded T2 cells were enumerated by FACS and expressed as %specific lysis in comparison to an internal control population oflabeled non-WT1-presenting T2 cells (FIGS. 5A-5F). As shown, each of theWT1-specific TCRs was able to specifically lyse WT-1 presenting targetcells.

For the cytokine expression study, IFNγ secretion was assessed (FIGS.6A-6F). Endogenous, presentation-deficient T2 cells were incubated withthe indicated concentrations of WT1 peptide, then incubated 16 hourswith polyclonal T cells expressing either no exogenous TCR (FIG. 6 ,polyclonal T cells), or expressing the indicated WT1-specific TCR. IFNγsecretion was assessed after 16 hours (FIGS. 6A-6F). As shown, each ofthe WT1-specific TCRs was able to induce IFNγ production afterincubation with WT-1 presenting target cells.

Additional cytolysis assays were performed to determine whether the WT1TCRs are capable of lysing target cells expressing endogenous WT1. FIG.7 provides the results of a further cytolysis assay for eJH52_13. U266cells were left un-manipulated (FIG. 7 , “endogenous expression”) orincubated with WT1 peptide (FIG. 7 , “+ peptide”), or incubated with theblocking anti-HLA-A2 antibody BB7.2 (FIG. 7 “+BB7.2 antibody). U266cells were then incubated for 16 hours with T cells expressing theeJH52_13 TCR. The loss of viable U266 cells was measured by FACS andexpressed as % specific lysis in relation to an internal controlpopulation of non-WT1-presenting cells. As shown in FIG. 7 , T cellsexpressing eJH52_13 (referred to in the figure as eJH52_WT1C_13) werecapable of lysing U266 cells, even when only presenting endogenouslevels of WT1. This lysis was MHC-specific as HLA-A2 antibody blockadeprevented cell lysis. FIG. 8 provides the results of a similar cytolysisassay for eJH30_7, eJH30_8, eJH52_13, eJH64_6, and eJH64_9. HLA-A2⁺K-562 cells were left un-manipulated (FIG. 8 “endogenous expression”) orincubated with WT1 peptide (FIG. 8 , “+ peptide”). K-562 cells were thenincubated 16 hours with T cells expressing the indicated TCRs. The lossof viable K-562 cells was measured by FACS and expressed as % specificlysis in relation to an internal control population ofnon-WT1-presenting cells. T cells expressing either eJH30_7(eJH30_WT1C_7), eJH52_13 (eJH52_WT1C_13), or eJH64_9 (eJH64_WT1C_9) wereall capable of lysing target cells expressing endogenous levels of WT1.

Similarly, eJH52_13 was capable of eliciting IFNγ expression in responseto endogenous WT1. FIG. 9 show the results of the study. HLA-A2⁺ K-562cells were left untreated (FIG. 9 , “endogenous expression”), incubatedwith WT1 peptide (FIG. 9 “+ peptide”), or transfected withWT1-epitope-containing in vitro transcribed RNA (ivtRNA) (FIG. 9 ,“forced overexpression”). Polyclonal or TCR-overexpressing T cells wereincubated with the indicated K-562 cells and assayed for IFNγ secretionafter 16 hours. eJH52_13-expressing T cells secreted appreciable amountsof IFNγ even when K-562 cells expressed endogenous levels of WT1 (FIG. 9, bottom panel).

A functional avidity assay was carried out to assess the potency of WT1TCRs eJH30_7, eJH52_13, eJH64_6, eJH64_9. The results are provided inFIGS. 10A-10D and FIG. 11 , and Table 5. TCR-deficient cells wereelectroporated with vectors encoding the indicated TCRs and allowed torest overnight. TAP-I-deficient T2 cells were pulsed with the indicatedconcentrations of WT1 10 mer peptide (YMLDLQPETT; SEQ ID NO: 97) for 30minutes, washed, and then mixed with the cells expressing the indicatedTCRs for 4 hours. Cells were stained for CD3 (as a measure of TCRexpression) and CD69, then analyzed by FACS. CD69 MFI of the TCR⁺ cellswas plotted against peptide concentration to assess the TCRs' relativepotency. FIG. 10A provides the potency of eJH30_7. FIG. 10B provides thepotency of eJH52_13. FIG. 10C provides the potency of eJH64_6. FIG. 10Dprovides the potency of eJH64_9. FIG. 11 and Table 5 show the relativepotencies of each TCR.

TABLE 5 EC₅₀ of functional avidity for WT1 TCRs TCR ID EC₅₀ eJH30_70.3646 eJH52_13 2.778 eJH64_6 0.7003 eJH64_9 0.8948

EMBODIMENTS

The following are exemplary enumerated embodiments of the presentdisclosure.

Embodiment 1. A recombinant T cell receptor (TCR) that binds Wilms'tumor antigen-1 (WT1), comprising an alpha chain and a beta chain,wherein the beta chain comprises a CDR3 sequence comprising an aminoacid sequence set forth in SEQ ID NOs: 22 or 30.Embodiment 2. A recombinant TCR that binds WT1, comprising an alphachain and a beta chain, wherein the alpha chain comprises a CDR3sequence set forth in SEQ ID NOs: 19 or 27.Embodiment 3. The recombinant TCR of Embodiment 1 or 2, wherein the betachain comprises a CDR2 sequence comprising an amino acid sequence setforth in SEQ ID NOs: 21 or 29.Embodiment 4. The recombinant TCR of any one of Embodiments 1-3, whereinthe alpha chain comprises a CDR2 sequence comprising an amino acid setforth in SEQ ID NOs: 18 or 26.Embodiment 5. The recombinant TCR of any one of Embodiments 1-4, whereinthe beta chain comprises a CDR1 sequence comprising an amino acidsequence set forth in SEQ ID NOs: 20 or 28.Embodiment 6. The recombinant TCR of any one of Embodiments 1-5, whereinthe alpha chain comprises a CDR1 sequence comprising an amino acidsequence set forth in SEQ ID NOs: 17 or 25.Embodiment 7. The recombinant TCR of Embodiment 1 or 2, wherein the TCRcomprises:

-   -   an alpha chain CDR1 comprising an amino acid sequence according        to SEQ ID NO: 17;    -   an alpha chain CDR2 comprising an amino acid sequence according        to SEQ ID NO: 18;    -   an alpha chain CDR3 comprising an amino acid sequence according        to SEQ ID NO: 19;    -   a beta chain CDR1 comprising an amino acid sequence according to        SEQ ID NO: 20;    -   a beta chain CDR2 comprising an amino acid sequence according to        SEQ ID NO: 21; and    -   a beta chain CDR3 comprising an amino acid sequence according to        SEQ ID NO: 22.        Embodiment 8. The recombinant TCR of Embodiment 1 or 2, wherein        the TCR comprises:    -   an alpha chain CDR1 comprising an amino acid sequence according        to SEQ ID NO: 25;    -   an alpha chain CDR2 comprising an amino acid sequence according        to SEQ ID NO: 26;    -   an alpha chain CDR3 comprising an amino acid sequence according        to SEQ ID NO: 27;    -   a beta chain CDR1 comprising an amino acid sequence according to        SEQ ID NO:28;    -   a beta chain CDR2 comprising an amino acid sequence according to        SEQ ID NO: 29; and    -   a beta chain CDR3 comprising an amino acid sequence according to        SEQ ID NO: 30.        Embodiment 9. The recombinant TCR of any one of Embodiments 1 to        8, wherein the amino acid sequence of the alpha chain TCR        comprises an amino acid sequence having at least 95% identity to        a sequence set forth in SEQ ID NOs: 23 or 31 and the beta chain        comprises an amino acid sequence having at least 95% identity to        a sequence set forth in SEQ ID NOs: 24 or 32.        Embodiment 10. The recombinant TCR of Embodiment 9, wherein:    -   a). the amino acid sequence of the alpha chain TCR comprises an        amino acid sequence having at least 95% identity to a sequence        set forth in SEQ ID NO: 23 and the beta chain comprises an amino        acid sequence having at least 95% identity to a sequence set        forth in SEQ ID NO: 24; or    -   b). the amino acid sequence of the alpha chain TCR comprises an        amino acid sequence having at least 95% identity to a sequence        set forth in SEQ ID NO: 31 and the beta chain comprises an amino        acid sequence having at least 95% identity to a sequence set        forth in SEQ ID NO:32.        Embodiment 11. The recombinant TCR of Embodiment 9 or 10,        wherein:    -   a). the amino acid sequence of the alpha chain TCR comprises an        amino acid sequence set forth in SEQ ID NO: 23 and the beta        chain comprises an amino acid sequence set forth in SEQ ID NO:        24; or    -   b). the amino acid sequence of the alpha chain TCR comprises an        amino acid sequence set forth in SEQ ID NO: 31 and the beta        chain comprises an amino acid sequence set forth in SEQ ID        NO:32.        Embodiment 12. The recombinant TCR of any one of Embodiments 1        to 11, wherein the TCR is capable of binding to an epitope on        the WT1 protein epitope or capable of eliciting a functional T        cell response.        Embodiment 13. The recombinant TCR of Embodiment 12, wherein        said T cell response is measured by CD69 expression or T cell        cytolysis.        Embodiment 14. The recombinant TCR of Embodiment 12 or 13,        wherein the epitope comprises a sequence according to SEQ ID NO:        97.        Embodiment 15. The recombinant TCR of any one of Embodiments 1        to 14, wherein the TCR is capable of binding to a WT1/HLA-A2        complex with an interaction half-life (t_(1/2)) of less than 100        seconds, or about 30 seconds to about 1000 seconds.        Embodiment 16. The recombinant TCR of any one of Embodiments 1        to 15, wherein the TCR binds a WT 1/HLA-A2 complex and is        capable of activating a functional T cell response and having an        EC₅₀ less than about 100 nM, less than about 10 nM, less than        about 1 nM, less than about 500 pM, less than about 100 pM, or        less than about 1 pM.        Embodiment 17. The recombinant TCR of any one of Embodiments 1        to 16, wherein the TCR is a soluble TCR.        Embodiment 18. The recombinant TCR of any one of Embodiments 1        to 17, wherein the TCR is coupled to an antibody or fragment        thereof.        Embodiment 19. The recombinant TCR of Embodiment 18, wherein the        antibody or fragment thereof is an anti-CD3 scFv or an anti-CD3        Fab.        Embodiment 20. A method for treating cancer in a subject in need        thereof, comprising administering to the subject an effective        amount of a composition comprising the recombinant TCR of any        one of Embodiments 1 to 19.        Embodiment 21. The method of Embodiment 20, wherein the cancer        is a leukemia.        Embodiment 22. A recombinant host cell comprising an exogenous        TCR of any one of Embodiments 1-21.        Embodiment 23. The recombinant host cell of Embodiment 22,        wherein the host cell is an immune cell.        Embodiment 24. The recombinant host cell of Embodiment 23,        wherein the immune cell is a T cell, NK cell, or NK T cell.        Embodiment 25. The recombinant host cell of Embodiment 24,        wherein the T cell is a naïve T cell, an effector T cell, a        central memory T cell, an effector memory T cell, a CD4⁺ T cell,        a CD8⁺ T cell, an alpha/beta T cell, a gamma/delta T cell, or        any combination thereof.        Embodiment 26. A method for treating cancer in a subject in need        thereof, comprising administering to the subject a recombinant        host cell according to any one of Embodiments 22-25.        Embodiment 27. A recombinant T cell receptor (TCR) that binds        Wilms' tumor antigen-1 (WT1), comprising an alpha chain and a        beta chain, wherein the beta chain comprises a CDR3 sequence        comprising an amino acid sequence

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1.-19. (canceled)
 20. A recombinant T cell receptor (TCR) that bindsWilms' tumor antigen-1 (WT1), comprising an alpha chain and a betachain, wherein the beta chain comprises a CDR3 sequence comprising theamino acid sequence set forth in SEQ ID NO: 6, 14, 22, 30, 38, or 46.21. The recombinant TCR of claim 20, wherein the beta chain comprises aCDR3 sequence comprising the amino acid sequence set forth in SEQ ID NO:22.
 22. The recombinant TCR of claim 21, wherein the alpha chaincomprises a CDR3 sequence comprising an amino acid sequence having atleast 90% sequence identity to an amino acid sequence set forth in SEQID NO:
 19. 23. The recombinant TCR of claim 22, wherein the alpha chaincomprises a CDR3 sequence comprising the amino acid sequence set forthin SEQ ID NO:
 19. 24. The recombinant TCR of claim 20, wherein the betachain comprises a CDR3 sequence comprising the amino acid sequence setforth in SEQ ID NO:
 30. 25. The recombinant TCR of claim 24, wherein thealpha chain comprises a CDR3 sequence comprising an amino acid sequencehaving at least 90% sequence identity to an amino acid sequence setforth in SEQ ID NO:
 27. 26. The recombinant TCR of claim 25, wherein thealpha chain comprises a CDR3 sequence comprising the amino acid sequenceset forth in SEQ ID NO:
 27. 27. The recombinant TCR of claim 20, whereinthe beta chain comprises a CDR3 sequence comprising the amino acidsequence set forth in SEQ ID NO:
 38. 28. The recombinant TCR of claim27, wherein the alpha chain comprises a CDR3 sequence comprising anamino acid sequence having at least 90% sequence identity to an aminoacid sequence set forth in SEQ ID NO:
 35. 29. The recombinant TCR ofclaim 28, wherein the alpha chain comprises a CDR3 sequence comprisingthe amino acid sequence set forth in SEQ ID NO:
 35. 30. A recombinantTCR that binds WT1, comprising an alpha chain and a beta chain, wherein:the alpha chain comprises a CDR3 sequence comprising an amino acidsequence having at least 90% sequence identity to an amino acid sequenceset forth in SEQ ID NO: 19; and the beta chain comprises a CDR3 sequencecomprising an amino acid sequence having at least 90% sequence identityto an amino acid sequence set forth in SEQ ID NO:
 22. 31. A recombinantTCR that binds WT1, comprising an alpha chain and a beta chain, wherein:the alpha chain comprises a CDR3 sequence comprising an amino acidsequence having at least 90% sequence identity to an amino acid sequenceset forth in SEQ ID NO: 27; and the beta chain comprises a CDR3 sequencecomprising an amino acid sequence having at least 90% sequence identityto an amino acid sequence set forth in SEQ ID NO: 30, or wherein: thealpha chain comprises a CDR3 sequence comprising an amino acid sequencehaving at least 90% sequence identity to an amino acid sequence setforth in SEQ ID NO: 27; and the beta chain comprises a CDR3 sequencecomprising an amino acid sequence having at least 90% sequence identityto an amino acid sequence set forth in SEQ ID NO:
 30. 32. Apharmaceutical composition comprising the recombinant TCR of claim 20.33. A pharmaceutical composition comprising the recombinant TCR of claim21.
 34. A pharmaceutical composition comprising the recombinant TCR ofclaim
 30. 35. A nucleic acid encoding the beta chain of the recombinantTCR of claim
 20. 36. A recombinant host cell comprising the nucleic acidof claim 35 or an expression vector comprising the nucleic acid operablylinked to an expression control sequence.
 37. A nucleic acid encodingthe beta chain of the recombinant TCR of claim
 21. 38. A recombinanthost cell comprising the nucleic acid of claim 37 or an expressionvector comprising the nucleic acid operably linked to an expressioncontrol sequence.